plants

Review
Citrus limon (Lemon) Phenomenon—A Review of the
Chemistry, Pharmacological Properties, Applications
in the Modern Pharmaceutical, Food, and Cosmetics
Industries, and Biotechnological Studies
Marta Klimek-Szczykutowicz, Agnieszka Szopa * and Halina Ekiert
Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, Medyczna 9,
30-688 Kraków, Poland; marta.klimek-szczykutowicz@doctoral.uj.edu.pl (M.K.-S.); mfekiert@cyf-kr.edu.pl (H.E.)
* Correspondence: a.szopa@uj.edu.pl; Tel.: +48-12-620-54-30




Received: 15 December 2019; Accepted: 14 January 2020; Published: 17 January 2020


Abstract: This review presents important botanical, chemical and pharmacological characteristics
of Citrus limon (lemon)—a species with valuable pharmaceutical, cosmetic and culinary (healthy
food) properties. A short description of the genus Citrus is followed by information on the chemical
composition, metabolomic studies and biological activities of the main raw materials obtained from
C. limon (fruit extract, juice, essential oil). The valuable biological activity of C. limon is determined
by its high content of phenolic compounds, mainly flavonoids (e.g., diosmin, hesperidin, limocitrin)
and phenolic acids (e.g., ferulic, synapic, p-hydroxybenzoic acids). The essential oil is rich in
bioactive monoterpenoids such as D-limonene, β-pinene, γ-terpinene. Recently scientifically proven
therapeutic activities of C. limon include anti-inflammatory, antimicrobial, anticancer and antiparasitic
activities. The review pays particular attention, with references to published scientific research, to the
use of C. limon in the food industry and cosmetology. It also addresses the safety of use and potential
phototoxicity of the raw materials. Lastly, the review emphasizes the significance of biotechnological
studies on C. limon.

Keywords: lemon; chemical composition; biological activity; cosmetic applications; phototoxicity;

biotechnological studies

1. Introduction
Citrus limon (L.) Burm. f. is a tree with evergreen leaves and yellow edible fruits from the family
Rutaceae. In some languages, C. limon is known as lemon (English), Zitrone (German), le citron (French),
limón (Spanish), and níngméng, 檸檬 (Chinese).
The main raw material of C. limon is the fruit, particularly the essential oil and juice obtained from
it. The C. limon fruit stands out as having well-known nutritional properties, but it is worth remarking
that its valuable biological activities are underestimated in modern phytotherapy and cosmetology [1].
C. limon fruit juice (lemon juice) has traditionally been used as a remedy for scurvy before the
discovery of vitamin C [2]. This common use of C. limon, known since ancient times, has nowadays
been supported by numerous scientific studies. Other uses for lemon juice, known from traditional
medicine, include treatment of high blood pressure, the common cold, and irregular menstruation.
Moreover, the essential oil of C. limon is a known remedy for coughs [3–5].
In Romanian traditional medicine, C. limon essential oil was administered on sugar for suppressing
coughs [3]. Aside from being rich in vitamin C, which assists in warding off infections, the juice is
traditionally used to treat scurvy, sore throats, fevers, rheumatism, high blood pressure, and chest
pain [6].

Plants 2020, 9, 119; doi:10.3390/plants9010119 www.mdpi.com/journal/plants

Plants 2020, 9, 119 2 of 24

In Trinidad, a mixture of lemon juice with alcohol or coconut oil has been used to treat fever,
coughs in the common cold, and high blood pressure. Moreover, the juice or grated skin, mixed with
molasses, has been used to remove excess water from the body, and the juice mixed with olive oil has
been administered for womb infection and kidney stones [4]. According to Indian traditional medicine,
C. limon juice can induce menstruation; the recommended dose for this is two teaspoons consumed
twice a day [5].
Currently, valuable scientific publications focus on the ever wider pharmacological actions of C.
limon fruit extract, juice and essential oil. They include studies of, for example, antibacterial, antifungal,
anti-inflammatory, anticancer, hepatoregenerating and cardioprotective activities [7–11].
The pharmacological potential of C. limon is determined by its rich chemical composition. The most
important group of secondary metabolites in the fruit includes flavonoids and also other compounds,
such as phenolic acids, coumarins, carboxylic acids, aminoacids and vitamins. The main compounds
of essential oil are monoterpenoids, especially D-limonene. These valuable chemical components are
the reason for the important position of C. limon in the food and cosmetics industries [12–14].
The aim of this overview is a systematic review of scientific works and in-depth analyses of
the latest investigations and promotions related to C. limon as a valuable plant species, important in
pharmacy, cosmetology and the food industry. Additionally, relevant biotechnological investigations
are presented.

2. The Genus Citrus

The genus Citrus is one of the most important taxonomic subunits of the family Rutaceae. Fruits
produced by the species belonging to this genus are called ‘citrus’ in colloquial language, or citrus
fruits. Citrus fruits are commonly known for their valuable nutritional, pharmaceutical and cosmetic
properties. The genus Citrus includes evergreen plants, shrubs or trees (from 3 to 15 m tall). Their leaves
are leathery, ovoid or elliptical in shape. Some of them have spikes. The flowers grow individually in
leaf axils. Each flower has five petals, white or reddish. The fruit is a hesperidium berry. The species
belonging to the genus Citrus occurs naturally in areas with a warm and mild climate, mainly in the
Mediterranean region. They are usually sensitive to frost [2].
One of the best known and most used species of the genus Citrus is the lemon—Citrus limon
(L.) Burm. f. (Latin synonyms: C. × limonia, C. limonum). Other important species included in this
taxonomic unit are: Citrus aurantium ssp. aurantium—bitter orange, Citrus sinensis—Chinese orange,
Citrus reticulata—mandarin, Citrus paradise—grapefruit, Citrus bergamia—bergamot orange, Citrus
medica—citron, and many others. A team of scientists from the University of California (Oakland,
California, USA) [15] analyzed the origin of several species of the genus Citrus, including C. limon.
They found that C. limon was a plant that had formed as a result of the combination of two species—C.
aurantium and C. medica. In the studies of scientists from Southwest University of China (Chongqing,
China), the metabolite profiles of C. limon, C. aurantium and C. medica were evaluated using gas
chromatography–mass spectrometry (GC-MS) and the partial least squares discriminant analysis
(PLS-DA) score plot [16]. They proved that C. limon has a smaller distance between C. aurantium and C.
medica in comparison with other Citrus species. These studies demonstrated that C. limon was likely a
hybrid of C. medica and C. aurantium, as previously suspected [16].
Botanical classification of the species of the genus Citrus is very difficult due to the frequent
formation of hybrids and the introduction of numerous cultivars through cross-pollination. Hybrids are
produced to obtain fruit with valuable organoleptic and industrial properties, including seedless fruit,
high juiciness, and the required taste. For older varieties, hybrids and cultivars, the latest molecular
techniques are often needed to identify them. C. limon, like many other prolific citrus species, gives
rise to numerous varieties, cultivars and hybrids, which are presented in Tables 1 and 2 acc. to [17].
Plants 2020, 9, 119 3 of 24

Table 1. C. limon cultivars.

Cultivar Name Origin Cultivation Characteristic

C. limon ‘Bearss’ (C. limon It grows quickly and is very productive. It has aromatic
Florida Florida, Brazil
‘Sicilian’, Bearss lemon) flowers, juicy fruit and a high sensitivity to low temperatures.
The specimens are large, without spines. It bloom two to
C. limon ‘Berna’—(C. limon three times a year. Fruits from individual harvests differ in
Spain Spain
‘Verna’, Verma lemon) properties and are called ‘Cosecha’ (main collections),
‘Secundus’ and ‘Rodrejos’.
C. limon ‘Eureka’ (Eureka Mediterranean Basin, California, Oblong fruit with a smooth skin and a small amount of
California, Sicily
lemon) Australia, Argentina, South Africa, Israel stones. Flowers of a pink shade.
A very productive variety. It blooms and bears fruit
C. limon ‘Femminello’ Italy Italy
throughout the year.
C. limon ‘Genova’ (C. limon Spike-free trees, resistant to cold with dense foliage. Yellow
Italy California, Florida, Chile
‘Genoa’) fruits with a marked tip have a smooth and thin pericarp.
It has large, oblong, cylindrical pointed fruit. Pericarp
C. limon ‘Interdonato’ Italy Italy strongly adheres to the fruit; it is thin, smooth, and yellow.
With few seeds.
California, Arizona, Australia, Uruguay, It has long spines, thick skin, pink flowers, and pale-yellow
C. limon ‘Lisbon’ Portugal
Argentina flesh.
The main advantage of this variety is high resistance to the
C. limon ‘Monachello’ Italy Italy
disease caused by Phoma tracheiphila.
C. limon ‘Primofiori’ (C. limon
A productive variety with spines. Fruits have a spherical or
‘Fino’, C. limon ‘Mesero’, C. Spain Spain
oval shape, with a small wart at the end.
limon ‘Blanco’
C. limon ‘Santa Teresa’ (C. limon Pericarp, contains a large amount of essential oil. The fruit
‘Feminello Santa Teresa’, C. Italy Italy, North-West Argentina, Turkey contains a large amount of juice. This variety is resistant to
limon ‘Italian’) storage and transport.
C. limon var. Variegata (C. limon Established as a result of the intrinsic mutation of C. limon
‘Eureka’ var. Variegated, California California ‘Eureka’ in 1931. It has pulp and juice of a pink shade. The
Pink-fleshed lemon fruits are yellow with green stripes and variegated leaves.
It has pulp and juice of a pink shade. The fruit is yellow with
C. limon ‘Villafranca’ Sicily Florida, Israel, North-West Argentina
green stripes.
Plants 2020, 9, 119 4 of 24

One of the oldest preserved botanical sources describing species of the genus Citrus is the
“Monograph on the Oranges of Wên-chou” (in Chinese: 記 嘉 桔 錄, “Citrus records of Ji Jia”) by
Han Yanzhi from 1178 [18,19]. Other historical works describing the species bearing citrus fruits are
“Nürnbergische Hesperides” from 1708 and “Traité du Citrus” from 1811. Historically, one of the best
known classifications of citrus species is “Histoire Naturelle des Orangers” from 1818. The American
botanist Walter Tennyson Swingle (1871–1952) had a particularly significant impact on the present-day
taxonomy of the genus Citrus. He is the author of as many as 95 botanical names of species of the genus
Citrus. Currently, the systematics of the species of the genus Citrus are based on studies of molecular
markers and other DNA analysis technologies still provide new information [20].

Table 2. Hybrids of C. limon.

Name Origin Characteristic

C. limon
hybrid C. limon and C. aurantifolia It has fruit larger than limes (C. aurantifolia).
‘Lemonime’
hybrid C. maxima and C. medica, The fruit resembles a pear. It can reach
C. limon ‘Lumia’
subsequently hybridized with C. limon large sizes.
C. limon
hybrid C. limon and C. medica Fruits with a pear-shaped and thick pericarp.
‘Ponderosa’
Specimens smaller than C. limon. The fruit has
few seeds and a thick, rough, light reddish
C. limon ‘Volkamer’ hybrid C. limon and C.aurantium
pericarp. The flesh and juice are yellow-red.
The hybrid is resistant to many diseases.

3. Botanical Characteristics and Occurrence of C. limon

Citrus limon (L.) Burm. f. (lemon) is a tree reaching 2.5–3 m in height. It has evergreen lanceolate
leaves. Bisexual flowers are white with a purple tinge at the edges of the petals. They are gathered
in small clusters or occur individually, growing in leaf axils. The fruit is an elongated, oval, pointed
green berry that turns yellow during ripening. Inside, the berry is filled with a juicy pulp divided
into segments (like an orange). The C. limon pericarp is made of a thin, wax-covered exocarp, under
which there is the outer part of the mesocarp, also known as flavedo. This part contains oil vesicles
and carotenoid dyes. The inner part of the mesocarp, also known as the albedo, is made of a spongy,
white parenchyma tissue. The endocarp, or ‘fruit flesh’, is divided into segments by the spongy, white
tissue of the mesocarp [2].
The C. limon tree prefers sunny places. It grows on loamy, well-drained, moist soils with a wide
pH range [1,2].
The location of the original natural habitat of C. limon is not accurately known [1,21]. However,
C. limon is considered to be native to North-Western or North-Eastern India [2,17].
C. limon is mainly recognized as a cultivated species. It has been cultivated in southern Italy since
the 3rd century AD, and in Iraq and Egypt since 700 AD. The Arabs introduced C. limon into Spain,
where it has been cultivated since 1150. Marco Polo’s expeditions also brought C. limon to China in
1297. It was also one of the first new species that Christopher Columbus brought in the form of seeds
to the North American continent in 1493. In the 19th century, worldwide commercial production of C.
limon began in Florida and in California. Nowadays, the USA is the largest producer of C. limon. Italy,
Spain, Argentina and Brazil also play a significant role [17].

4. C. limon Pharmacopoeial Monographs and Safety of Use

By cold-pressing the fresh outer parts of the C. limon pericarp (Lat. exocarpium), an essential oil
is obtained—the lemon oil (lat. Citrus limon aetheroleum, Limonis aetheroleum, Oleum Citri). The oil is
colourless or yellow, and has a characteristic, strong lemon scent [21]. It is considered a pharmacopoeial
Plants 2020, 9, 119 5 of 24

raw material. Its monographs, entitled ‘Limonis aetheroleum’, are present in the European Pharmacopoeia
9th [22], American Pharmacopoeia [23], and in the Ayurvedic Pharmacopoeia of India [24].
Another pharmacopoeial raw material obtained from C. limon is the outer part of the mesocarp—the
flavedo. A monograph entitled ‘Citrus limon flavedo’ can be found in older editions of the French
Pharmacopoeia, for example, in its 10th edition from 1998 [25].
The fresh fruit of C. limon is officially listed for use in phytotherapy and in homeopathy in Germany.
According to the German Commission D Monographs for homeopathic medicines, C. limon fresh fruits
can be used for treating gingival bleeding and debilitating diseases [26].
C. limon also has a positive recommendation in the European Commission’s Cosmetics Ingredients
Database (CosIng Database) as a valuable plant for cosmetics’ production [27].
The European Food Safety Authority (EFSA) classified the pericarp, fruit, and leaves of C. limon
as raw materials of plant origin, in which there is presence of naturally occurring ingredients that
may pose a threat to human health when used in the production of food and dietary supplements.
EFSA has remarked that the toxic substances in these raw materials are photosensitizing compounds
belonging to the furanocoumarin group, including bergapten and oxypeucedanin (Figure 1) [28].

Figure 1. Chemical structure of selected linear furanocoumarins, determining the photosensitizing

effect of C. limon.

In the American Food and Drug Administration (FDA) list, C. limon essential oil and extracts are
classified as safe products [29].

5. Chemical Composition of C. limon

The chemical composition of C. limon fruit is well known. It has not only been determined for the
whole fruit but also separately for the pericarp, juice, pomace, and essential oil. The compositions of
the leaves and the fatty oil extracted from C. limon seeds are also known. Due to the large number of C.
limon varieties, cultivars and hybrids, various research centres undertake the task of analyzing the
chemical composition of the raw materials obtained from them.
The most important group of bioactive compounds in both C. limon fruit and its juice, determining
their biological activity, are flavonoids such as: flavonones—eriodictyol, hesperidin, hesperetin,
naringin; flavones—apigenin, diosmin; flavonols—quercetin; and their derivatives (Figure 2). In the
whole fruit, other flavonoids are additionally detected: flavonols—limocitrin (Figure 2) and spinacetin,
and flavones—orientin and vitexin (Tables 3 and 4). Some flavonoids, such as neohesperidin, naringin
and hesperidin (Figure 2), are characteristic for C. limon fruit. In comparison to another Citrus species,
C. limon has the highest content of eriocitrin (Figure 2) [30].
Plants 2020, 9, 119 6 of 24

Figure 2. Chemical structure of flavonoids characteristic of C. limon.

Phenolic acids are another important group of compounds found both in the juice and fruit.
There are mainly two such compounds in the juice—ferulic acid and synapic acid, and their derivatives.
In contrast, the presence of p-hydroxybenzoic acid has been confirmed in the fruit. In the fruit, there
are also coumarin compounds, carboxylic acids, carbohydrates, as well as amino acids, a complex of B
vitamins, and, importantly, vitamin C (ascorbic acid) (Tables 3 and 4) [1,12,13,31–36].
Plants 2020, 9, 119 7 of 24

Table 3. Composition of C. limon fruits extracts.

Group of
Part of Fruit Metabolites
Compounds
flavonones: eriocitrin, eriodiktyol, hesperidin, naringin,
neoeriocitrin, neohesperidin
Whole fruit (pulp, flavones: apigenin, diosmetin, diosmin, homoorientin,
Flavonoids
seed and peel) luteolin, orientin, vitexin
flavonols: isoramnethin, quercetin, limocitrin, rutoside,
spinacetin
Whole fruit (pulp,
Limonoids limonin, nomilin
seed and peel)
Whole fruit (pulp, dihydroferulic acid, p-hydroxybenzoic acid,
Phenolic acids
seed and peel) 3-(2-hydroxy-4-methoxyphenyl)propanoic acid, synapic acid
citric acid, galacturonic acid, glucuronic acid, glutaric acid,
Whole fruit (pulp,
Carboxylic acids homocitric acid, 3-hydroxymethylglutaric acid, isocitric acid,
seed and peel)
malic acid, quinic acid
Whole fruit (pulp,
Coumarins citropten (5,7-dimethoxycoumarin), scopoletin
seed and peel)
Whole fruit (pulp,
Furanocoumarins bergamottin
seed and peel)
L-alanine, L-arginine, L-asparagine, L-aspartic acid,
Whole fruit (pulp,
Amino acids dimethylglycine, glutamic acid, L-phenylalanine,
seed and peel)
DL-proline, L-tryptophan, L-tyrosine, L-valine
monosaccharides: arabinose, fructose, β-fructofuranose,
Peel β-fructopyranose, galactose, glucose, mannose, myoinositol,
rhamnose, scylloinositol, xylose
Carbohydrates
Whole fruit (pulp,
disaccharides: sucrose
seed and peel)
Vitamins and Whole fruit (pulp,
choline, pantothenic acid, trigoneline, vitamin C
theirsmetabolites seed and peel)
calcium (Ca), magnesium (Mg), phosphorus (P), potassium
Macroelements Pulp and peel
(K), sodium (Na)

Table 4. Composition of C. limon juice.

Group of Compounds Metabolites

flavonones: hesperidin, naringin
flavones: apigenin, chrysoeriol, diosmetin, luteolin
Flavonoids
flavonols: isoramnethin, quercetin, rutoside
dihydroxyflavonols: dihydroxyisoramnethin-7-O-rutinoside
Phenolic acids ferulic acid, synapic acid
Vitamins vitamins: C (53 mg/L), A, B1 , B2 , B3

Another interesting group of compounds that are found in C. limon fruits are limonoids. Limonoids
are highly oxidized secondary metabolites with polycyclic triterpenoid backbones. They mainly occur
in citrus fruits, including lemons, in which they are found mainly in the seeds, pulp, and peel. There are
predominantly two such compounds in C. limon fruits—limonin and nomilin (Figure 3) [37]. Studies
have shown that the concentrations of the compounds of this group are dependent on fruit growth and
maturation stages. Young citrus fruits contain the highest amounts of these compounds, compared to
ripe ones [38].
Plants 2020, 9, 119 8 of 24

Figure 3. Chemical structure of limonoids characteristic of C. limon.

Analysis of macroelements in C. limon fruit showed the presence in pulp and peel of: calcium
(Ca), magnesium (Mg), phosphorus (P), potassium (K) and sodium (Na) [36].
In C. limon seed oil, the main ingredients are fatty acids, such as arachidonic acid, behenic acid
and linoleic acid, and also tocopherols and carotenoids (Table 5) [33,35]. The latest studies showed that
C. limon fruit pulp oil contains more fatty acids compared to other Citrus species, such as C. aurantium,
C. reticulata and C. sinensis. The following fatty acids have been identified in C. limon pulp oil: behenic
acid, erucic acid, gondoic acid, lauric acid, linoleic acid, α-linolenic acid, margaric acid, palmitic acid,
palmitoleic acid, pentadecanoic acid, and stearic acid [39].

Table 5. Composition of oil from C. limon seeds.

Group of Compounds Metabolites

arachidonic acid, behenic acid, lignoceric acid, linoleic acid, linolenic acid, oleic
Fatty acids
acid, oleopalmitic acid, palmitic acid, stearic acid
Tocopherols α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol
Carotenoids β-carotene, β-cryptoxanthin, lutein

The main components of the C. limon essential oil are monoterpenoids. Among them, quantitatively
dominant in the essential oil obtained from pericarp are: limonene (69.9%), β-pinene (11.2%),
γ-terpinene (8.21%), (Figure 4), sabinene (3.9%), myrcene (3.1%), geranial (E-citral, 2.9%), neral
(Z-citral, 1.5%), linalool (1.41%). In addition to terpenoids, the essential oil also contains linear
furanocoumarins (psoralens) and polymethoxylated flavones (Table 6) [14,40,41].

Figure 4. Chemical structure of selected terpenoids characteristic of C. limon essential oil.

Plants 2020, 9, 119 9 of 24

Table 6. The chemical composition of the essential oil of the C. limon pericarp and leaf.

Group of
Essential Oil Metabolites
Compounds
limonene (69.9%), p-mentha-3,8-diene (18.0%), β-pinene
(11.2%), γ-terpinene (8.21%), myrcene (4.4%), sabinene
(3.9%), myrcene (3.1%) geranial (E-citral, 2.9%), neral
(Z-citral, 1.5%), linalool (1.41%), α-pinene (1.1%), α-thujene
(1.1%), β-bisabolene (0.5%) (E)-β-ocimene (0.4%), citronellol
(0.3%), geraniol (0.2%), β-caryophyllene (0.2%),
essential oil of the
trans-muurala-4(14),5-diene (0.2%), α-terpinene (0.1%),
C. limon pericarp
terpinolene (0.1%), nonanal (0.1%), eucalyptol (0.1%); other
terpenes below 0.06%: α-bisabolol, camphene, citronellal,
citronellyl acetate, β-curcumene, γ-curcumene, p-cymene,
7-epi-sesquithujene, α-farnesene, α-felandren, cis-limonene,
Terpenoids trans-limonene, octanal, octanal acetate, terpinen-4-ol,
β-santalene, zonarene
limonene (31.5%), sabinene (15.9%), citronellal (11.6%),
linalool (4.6%), neral (4.5%), geranial (4.5%), (E)-β-ocimene
(3.9%), myrcene (2.9%), citronellol (2.3%), β-caryophyllene
(1.7%), terpne-4-ol (1.4%), geraniol (1.3%), α-pinene
(1.2%),γ-terpinene (0.9%), sylvestrene (0.6%), α-terpineol
essential oil of the
(0.6%), isogeranial (0.4%), β-bisabolene (0.3%), germacrene B
C. limon leaf
(0.3%), isospathulenol (0.3%), α-terpinene (0.2%), terpinolene
(0.2%), isopulegol (0.2%), γ-terpineol (0.2%), decanal (0.2%),
δ-elemene (0.2%), α-humulene (0.2%), α-cadinol (0.2%),
epi-α-bisabolol (0.2%) cis-p-menth-2-en-1-ol (0.1%), isoneral
(0.1%), γ-muurolene (0.1%), spathulenol (0.1%)
aprindine, bergamottin, bergapten, byakangelicol,
byakangelicin, epoxybergamottin, 5- and
essential oil of the
Furano-coumarins 8-geranoxypsoralen, 8-geranyloxypsoralen, heraclenin,
C. limon pericarp
imperatorin, isoimperatorin, isopimpinellin, xanthotoxin,
oxypucedanin, phellopterin, psoralen
essential oil of the citropten, 5-geranyloxy-7-methoxycoumarin, herniarin,
Coumarins
C. limon pericarp 5-isopentenyloxy-7-methoxycoumarin

The essential oil of the C. limon leaf differs in composition from oil obtained from pericarp. Its main
compounds include: limonene (31.5%), sabinene (15.9%), citronellal (11.6%), linalool (4.6%), neral
(4.5%), geranial (4.5%), (E)-β-ocimene (3.9%), myrcene (2.9%), citronellol (2.3%), β-caryophyllene
(1.7%), terpne-4-ol (1.4%), geraniol (1.3%) and α-pinene (1.2%) (Table 6) [14,16,40–43].

6. Metabolomic Profile Studies

The team of Mucci et al. [35] investigated the metabolic profile of different parts of C. limon
fruit. Flavedo, albedo, pulp, oil glands, and the seeds of lemon fruit and citron were studied through
high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy.
The analyses were made directly on intact tissues without any physicochemical manipulation.
In C. limon flavedo were detected: terpenoids (limonene, β-pinene and γ-terpinene), aminoacids
(asparagine, arginine, glutamine, proline), organic acids (malic acid and quinic acid), osmolites
(stachydrine), and fatty acid chains and sugars (glucose, fructose, β-fructofuranose, myoinositol,
scylloinositol and sucrose) (Table 3). The albedo of C. limon fruit showed the presence of low signals
from: aminoacids (alanine, threonine, valine, glutamine), sugars (glucose, sucrose, β-fructofuranose,
myoinosytol, scylloinositol and β-fructopyranose), and osmolites (stachydrine, β-hydroxybutyrate,
ethanol) (Table 3). In albedo, clear signals from flavonoids were detected, such as hesperidin and
rutoside, that have been identified also by high performance liquid chromatography (HPLC) analyses.
Oil glands’ HR-MAS NMR composition analysis showed the presence of terpenoids (limonene,
Plants 2020, 9, 119 10 of 24

γ-terpinene, β-pinene, α-pinene, geranial, neral, citronellal, myrcene, sabinene, α-thujene, nerol and
geraniol esters) and sugars (glucose, sucrose, β-fructofuranose and β-fructopyranose). The analysis
of C. limon pulp showed the presence of aminoacids (asparagine, proline, alanine, γ-aminobutyric
acid (GABA), glutamine, threonine and valine), organic acids (citric acid and malic acid), sugars
(glucose, sucrose, β-fructofuranose, β-fructopyranose, myoinosytol and scylloinosytol) and osmolites
(stachydrine, ethanol and methanol) (Table 3). HR-MAS NMR seeds analysis indicated that their
composition is dominated by triglyceride signals (linoleic acid, linolenic acid and their derivatives),
sugars (glucose and sucrose), osmolites (stachydrine) and trigonelline [35].
In another metabolomic study, the peel extracts of ripened C. limon fruit was characterized
as containing nonfluorescent chlorophyll catabolites (NCCs) and dioxobilane-type nonfluorescent
chlorophyll catabolite (DNCC) [44]. In the peels of C. limon fruit, four chlorophyll catabolites were
detected: Cl-NCC1, Cl-NCC2, Cl-NCC3 and Cl-NCC4 [44].
The metabolomic profile of C. limon leaf was investigated by Asai et al. [45]. The studies showed
that C. limon leaves contain 26 different organic acids and their derivatives (aconitic acid, 2-aminobutyric
acid, 4-aminobutyric acid, ascorbic acid, benzoic acid, citramalic acid, citric acid, p-coumaric acid,
ferulic acid, fumaric acid, glucaric acid, glycolic acid, 3-hydroxybutyric acid, 2-isopropylmalic acid,
malic acid, malonic acid, 3-methylglutaric acid, oxamic acid, D-3-phenyllacetic acid, pipecolic acid,
pyruvic acid, quinic acid, shikimic acid, succinic acid, threonic acid, urocanic acid), 21 aminoacids
(alanine, γ-aminobutyric acid, anthranilic acid, asparagine, aspartic acid, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamic acid,
serine, threonine, tryptophan, tyrosine, valine), and 13 sugars and sugar alcohols (arabinose, fructose,
galactose, glucose, glycerol, inositol, lyxose, maltose, rhamnose, ribose, sorbose, sucrose, xylitol).
Additionally, studied leaves have been exposed to stress conditions (leaves were placed in such a way
that the edge of the petiole was in contact with the bottom of a glass bottle, soaked with 0.2 mM jasmonic
acid and salicylic acid aqueous solutions, and incubated at 25 ◦ C for 24 h). The content of aminoacids,
such as, tyrosine, tryptophan, phenylalanine, valine, leucine, isoleucine, lysine, methionine, threonine,
histidine, and γ-aminobutyric acid, was increased after this stress treatment [45].
According to Mehl et al. [46], the identification of volatile and non-volatile metabolites in C. limon
essential oil is dependent on geographic origin and the analytical methods used. To evaluate the
potential of volatile and non-volatile fractions for classification purposes, volatile compounds of
cold-pressed lemon oils were analyzed, using modern methods like gas chromatography-flame
ionization detector-mass spectrometer (GC-FID/MS) and fourier transform mid-infrared spectroscopy
(FT-MIR), while the non-volatile residues were studied using FT-MIR with proton nuclear magnetic
resonance (1 H-NMR) and ultra-high performance liquid chromatography-quadrupole time-of-flight
mass spectrometry (UHPLC-TOF-MS). The studies lead to very good differentiation and classification
of samples regarding their geographic origin and extraction process modalities. The essential oil from
the Italian-originated C. limon fruit was enriched in α-thujene, α-pinene, α-terpinene, sesquiterpenoids
(i.e., β-caryophyllene) and furocoumarins (i.e., bergamottin). The essential oil from Spanish and
Argentinian C. limon fruit was characterized by significant terpene contents, such as limonene, but
differed in imperatorin, and byakangelicol contents. The studies showed that essential oil from Spanish
C. limon fruit contained more camphor and 4-terpineol, while Argentinian C. limon fruit contained
more sabinene and cis-sabinene hydrate [46].
The studies performed by Jing et al. [16] focused on the identification of components in the
essential oil of different Citrus species, including C. limon. In general, most of the studied essential oil
components were identified as monoterpenoids. The major monoterpenes in C. limon essential oil were:
limonene (70.37%), p-mentha-3,8-diene (18.00%), myrcene (4.40%), α-pinene (3.24%), α-thujene (1.05%)
and terpinolene (0.90%) (Table 6). Other monoterpenoids, which were identified as characteristic of C.
limon, were: sabinene (0.28%), α-terpinene (0.22%), trans-muurola-4(14), 5-diene (0.18%), eucalyptol
(0.12%), octanol acetate (0.03%), β-curcumene (0.03%), zonarene (0.03%), 7-epi-sesquithujene (0.02%),
citronellyl acetate (0.02%), α-farnesene (0.01%) (Table 6). The shown metabolite-based profiling
Plants 2020, 9, 119 11 of 24

model can be used to clearly discriminate the basic Citrus species. Limonene, α-pinene, sabinene
and α-terpinene were the major characteristic components of the analyzed metabolomes of Citrus
genotypes that contributed to their taxonomy [16].
Studies performed by Masson et al. [43] deal with furanocoumarin’s and coumarin’s metabolomic
profile in essential oil from C. limon fruit peel. C. limon essential oil contained large amounts of
both furanocoumarins and coumarins compared to another tested Citrus essential oils. In C. limon
essential oil, 13 furanocoumarins were detected (bergamottin, bergapten, byakangelicol, byakangelicin,
epoxybergamottin, 8-geranyloxypsoralen, heraclenin, imperatorin, isoimperatorin, isopimpinellin,
oxypeucedanin, oxypeucedanin hydrate, phellopterin) and two coumarins (citropten and herniarin)
(Table 6) [43].

7. Biological Activity of C. limon Raw Materials

7.1. Anticancer Activity

C. limon nanovesicles have been isolated from the fruit juice using the ultracentrifugation method
and purification on a 30% sucrose gradient, using an in vitro approach. The study showed that isolated
nanovesicles (20 µg/mL) inhibited cancer cell proliferation in different tumour cell lines, by activating a
TRAIL-mediated apoptotic cell death. Furthermore, C. limon nanovesicles suppress chronic myeloid
leukemia (CML) tumour growth in vivo by specifically reaching the tumour site and by activating
TRAIL-mediated apoptotic cell processes (Table 7) [47].
Another study has shown that an 80:20 methanol:water extract from lemon seeds induces apoptosis
in human breast adenocarcinoma (MCF-7) cells, leading to the inhibition of proliferation. This extract
showed the highest (29.1%) inhibition of MCF-7 cells in an MTT assay (Cell Proliferation Kit), compared
to ethyl acetate, acetone and methanol extracts. The results suggest that aglycones and glycosides of
the limonoids and flavonoids present in the 80:20 methanol:water extract may potentially serve as a
chemopreventive agent for breast cancer (Table 7) [9].

7.2. Antioxidant Activity

It has been shown that the antioxidant activity of the flavonoids from C. limon—hesperidin
and hesperetin—was not only limited to their radical scavenging activity but also augmented the
antioxidant cellular defences via the ERK/Nrf2 signalling pathway (Table 7) [8].
In addition, vitamin C prevents the formation of free radicals and protects DNA from mutations.
Studies have also shown a reduction in lipid peroxidation in seizures and status epilepticus was
induced by pilocarpine in adult rats [48].

7.3. Anti-Inflammatory Activity

Various in vitro and in vivo studies have been conducted to evaluate hesperidin metabolites, or
their synthetic derivatives, at their effectiveness in reducing inflammatory targets including NF-κB,
iNOS, and COX-2, and the markers of chronic inflammation (Table 7) [8].
The essential oil from C. limon (30 or 10 mg/kg p.o.) exhibited anti-inflammatory effects in
mice under formalin test by reducing cell migration, cytokine production and protein extravasation
induced by carrageenan. These effects were also obtained with similar amounts of pure D-limonene.
The anti-inflammatory effect of C. limon essential oil is probably due to the high concentration of
D-limonene (Table 8) [49].
Studies by Mahmoud et al. [50] have shown the protective effects of limonin on experimentally
induced hepatic ischemia reperfusion (I/R) injury in rats. The mechanism of these hepatoprotective
effects was related to the antioxidant and anti-inflammatory potential of limonin mediated by the
down-regulation of the TLR-signaling pathway [50].
In studies with the essential oil administered at a dose of 10 mg/kg p.o., D-limonene induced
a significant reduction in intestinal inflammatory scores, comparable to that induced by ibuprofen.
Plants 2020, 9, 119 12 of 24

The studies documented that D-limonene-fed rats had significantly lowered serum concentrations
of TNF-α compared to untreated TNBS-colitis rats. The anti-inflammatory effect of D-limonene also
involved the inhibition of TNFα-induced NF-κB translocation in fibroblast cultures. The application
of D-limonene in colonic HT-29/B6 cell monolayers increased epithelial resistance. The study found
evidence that IL-6 markedly decreased during dietary supplementation with D-limonene [51]. Another
study showed that the oil moderately inhibited soybean 5-lipoxygenase (5-LOX) with an IC50 value of
32.05 µg/mL (Table 8) [52].

7.4. Antimicrobial Activity

Acetone extracts from C. limon fruits have shown inhibitory activity against the Gram-positive
bacteria Enterococcus faecalis (MIC 0.01 mg/mL) and Bacillus subtilis (MIC 0.01 mg/mL), and the
Gram-negative Salmonella typhimurium (MIC 0.01 mg/mL) and Shigella sonnei (MIC 0.01 mg/mL)
(Table 7) [7].
Moreover, under another study, C. limon essential oil showed antibacterial activity against
Gram-positive bacteria (Bacillus subtilis (MIC 2 mg/mL), Staphylococcus capitis (MIC 4 mg/mL),
Micrococcus luteus (MIC 4 mg/mL)), and Gram-negative (Pseudomonas fluorescens (MIC 4 mg/mL),
Escherichia coli (100% inhibition)) (Table 8) [52,53].
The C. limon essential oil exhibits inhibitory activity against Staphylococcus mutans (MIC 4.5 mg/mL)
and effectively reduced the adherence of S. mutans on a glass surface, with adherence inhibition rates
(AIR) from 98.3% to 100%, and on a saliva-coated enamel surface, for which the AIRs were from 54.8%
to 79.2%. It effectively reduced the activity of glucosyltransferase (Gtf) and the transcription of Gtf in a
dose-dependent manner (Table 8) [54].
Ethanol and acetone extracts from fruits of C. limon were active against Candida glabrata (MIC
0.02 mg/mL) (Table 7) [7]. On the other hand, C. limon essential oil ingredients, such as D-limonene,
β-pinene and citral, have shown inhibitory activity against Aspergillus niger (MIC 90 µL/mL at 70
◦ C), Saccharomyces cerevisiae (MIC 4 mg/mL) and Candida parapsilosis (MIC 8 mg/mL) (Table 8) [52,55].

Another study confirmed that C. limon essential oil promoted a 100% reduction in the growth of C.
albicans [56].
Moreover, other studies have shown that C. limon essential oil at a concentration of 0.05% inhibits
Herpes simplex replication to the extent of 33.3% (Table 8) [57].

7.5. Antiparasitic Effect

The effect of C. limon essential oil on Sarcoptes scabiei var. cuniculi has been evaluated in vitro and
in vivo. The infected parts of rabbits were treated topically once a week for four successive weeks.
In vitro application results showed that C. limon essential oil (10% and 20%, diluted in water) caused
mortality in 100% of mites after 24 h post-application. In vivo application of 20% lemon oil on naturally
infected rabbits showed complete recovery from clinical signs and absence of mites in microscopic
examination from the second week of treatment (Table 8) [58].

7.6. Anti-Allergic Effect

Aqueous extracts from the peel of C. limon fruits have been used to investigate their effects on
the release of histamine from rat peritoneal exudate cells (PECs). The extracts inhibited the release of
histamine from rat PECs induced by the calcium ionophore A23187. Heating the extracts at 100 ◦ C for
10 min. enhanced the inhibition of histamine release. Histamine release was inhibited to the extent of
80%. The extracts potentially suppressed inflammation in mice cavity, like indometacin, a well-known
anti-inflammatory drug (Table 7) [59].

7.7. Hepatoregenerating Effect

An ethanolic extract of C. limon fruits has been evaluated for its effects on experimental liver
damage induced by carbon tetrachloride (CCl4 ), and the ethyl acetate soluble fraction of the extract has
Plants 2020, 9, 119 13 of 24

been evaluated for its effect on the HepG2 cell line (human liver cancer cell line). The ethanolic extract
(150 mg/mL) normalized the levels of aspartate aminotransferase (ASAT), alanine aminotransferase
(ALAT), alkaline phosphatase (ALP), and total direct bilirubin, which had been altered due to CCl4
intoxication in rats. After treatment with the extract, the level of malondialdehyde in the liver tissue
was significantly reduced, hence the lipid peroxidation, and raised the level of the antioxidant enzymes
superoxide dismutase and catalase. It improved the reduced glutathione levels in the treated rats
in comparison with CCl4 -intoxicated rats. The effect seen was dose dependent, and the effect of the
highest dose was almost equal to the standard—silymarin. In an investigation carried out on a human
liver-derived HepG2 cell line, a significant reduction in cell viability was observed in cells exposed to
CCl4 (Table 7) [10].
Studies with C. limon essential oil have also shown the stimulation of liver detoxification by the
activation of cytochrome P450 and liver enzymes (glutathione S-transferase) in chronic liver poisoning
(Table 8) [21].

7.8. Antidiabetic Effect

Ethanol extracts from C. limon peel were administered orally at a dose of 400 mg/kg daily for
12 days to diabetic rats in which diabetes had been induced by the use of streptozotocin. The study
showed a reduction in blood glucose, a reduction in wound healing time, and an increase in tissue
growth rate, collagen synthesis, and protein and hydroxyproline levels (Table 7) [60].
Another study evaluated the antidiabetic effect of D-limonene in streptozotocin-induced diabetic
rats. D-limonene was administered orally at doses of 50, 100 and 200 mg/kg body weight, and
glibenclamide at a dose of 600 µg/kg body weight, daily for 45 days. The administration of D-limonene
for 45 days gradually decreased the blood glucose level, and the maximum effect was observed at a dose
of 100 mg/kg body weight. The activities of gluconeogenic enzymes, such as glucose 6-phosphatase
and fructose 1,6-bisphosphatase, were increased, and the activity of the glycolytic enzyme, glucokinase,
was decreased, along with liver glycogen, in the diabetic rats. The effect of D-limonene was more
pronounced at the dose of 100 mg/kg body weight than at the two smaller doses. The antidiabetic
effect of D-limonene was comparable with that of glibenclamide (Table 8) [61].

7.9. Anti-Obesity Activity

In a study, lemon juice was used in a low-calorie diet (‘lemon detox diet’). The diet consisted of
2 L of lemon detox juice containing 140 g ‘Neera’ syrup, 140 g lemon juice, and 2 L water per day.
The study showed that C. limon juice caused a reduction in serum high-sensitive C-reactive protein
(hs-CRP) in comparison with the placebo and normal diet group. Haemoglobin and haematocrit levels
remained stable in the group on the lemon detox diet, while they decreased in the placebo and normal
diet groups (Table 7) [62].
Studies have shown that D-limonene is beneficial to people with dyslipidaemia and
hyperglycaemia. D-limonene at a dose of 400 mg/kg per day for 30 days promotes in male rats
a decrease in LDL-cholesterol, prevents the accumulation of lipids, and affects the blood sugar level.
Its antioxidant action enhances these effects. Dietary supplementation with D-limonene would restore
pathological alteration of the liver and pancreas. It could help in the prevention of obesity (Table 8) [21].

7.10. Effects on the Digestive System

Studies have shown that D-limonene increases gastric motility and causes a reduction in nausea,
neutralization of stomach acids, and relief of gastric reflux (Table 8) [21].

7.11. Effects on the Cardiovascular System

A study has indicated that daily intake of C. limon juice has a beneficial effect on blood pressure.
The study was conducted on 100 middle-aged women in an island area nearby Hiroshima. Instances of
lemon juice ingestion and the number of steps walked had been recorded for five months. The results
Plants 2020, 9, 119 14 of 24

indicated that daily lemon juice intake and walking were effective in reducing high blood pressure
because both showed significant negative correlations with systolic blood pressure (Table 7) [63].
In vitro and in vivo studies have confirmed that C. limon juice (0.4 mL/kg) has a significant
impact on blood pressure and on coagulation and anticoagulation factors in rabbits. In vitro tests
revealed a highly significant increase in thrombin time and activated partial thromboplastin time by C.
limon, whereas fibrinogen concentration was significantly reduced in comparison with the control;
prothrombin time, however, was not affected significantly. Significant changes were observed in
haematological parameters, such as amounts of erythrocytes and haemoglobin and mean corpuscular
haemoglobin concentrations, in in vivo testing of C. limon. Bleeding time and thrombin time were
significantly prolonged, and there was an increase in protein C and thrombin–antithrombin complex
levels (Table 7) [11].

7.12. Influence on the Nervous System

The influence of C. limon juice on the memory of mice has been investigated using Harvard
Panlab Passive Avoidance response apparatus, controlled through the LE2708 Programmer. Passive
Avoidance is a fear-motivated test used to assess the short- or long-term memory of small animals,
which measures the latency in entering a black compartment. Animals that were fed C. limon juice
(0.2, 0.4 and 0.6 mL/kg) showed, in comparison with the control, a highly significant or a significant
increase in latency before entering a black compartment after 3 and 24 h, respectively (Table 7) [64].
Studies have also shown that the main compound of C. limon essential oil—D-limonene—in
concentrations of 0.5% and 1.0%, administered to mice by inhalation, has a significant calming and
anxiolytic effect by activating serotonin and dopamine receptors. In addition, D-limonene has an
inhibitory effect on pain receptors, similar to that of indomethacin and hyoscine (Table 8) [65].

7.13. Influence on Skeletal System

Studies have shown the potential use of nomilin for the inhibition of osteoclastogenesis in vitro.
Cell viability of the mouse RAW264.7 macrophage cell line and mouse primary bone-marrow-derived
macrophages (BMMs) with the Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan) was
measured. Nomilin caused significantly decreased TRAP-positive multinucleated cell numbers
(a measure of osteoclast cell numbers) when compared with the control. Moreover, the
non-toxic concentrations of the compound decreased bone resorption activity and down regulated
osteoclast-specific genes (NFATc1 and TRAP mRNA levels), coupled with suppression of the MAPK
signaling pathway. Studies have shown the therapeutic potential of nomilin for the prevention of bone
metabolic diseases such as osteoporosis [66].

7.14. C. limon as Corrigent in Pharmacy

In addition to the very important uses mentioned above, the oil is used in pharmacy and cosmetic
formulations as a flavour and aroma corrigent, as well as a natural preservative, due to its confirmed
antibacterial and fungistatic effects [21].
Plants 2020, 9, 119 15 of 24

Table 7. Biological activity of C. limon fruit extracts confirmed by scientific research.

Activity Mechanism of Action References

- Inhibition of the proliferation of cancer cells;

- Activation of “TRAIL”-apoptopic cell death;
Anticancer activity - Inhibition of tumour growth in chronic yelogenous leukaemia (CML); [9,47]
- Antioxidant action and induction of apoptosis in MCF-7 cells (breast
adenocarcinoma cells) (C. limon seed extract).

- Augmention of antioxidant cellular defences via ERK/Nrf2

Antioxidant activity [8,48]
signalling pathway.

- Inhibition of NF-κB factor, nitric oxide (II) synthase (iNOS), induced

Anti-inflammatory activity cyclooxygenase (COX-2) (hesperidin, hesperitin); [8,49,50,52]
- Down-regulation of TLR-signaling pathway (limonin).

- Inhibiting activity against Gram-positive bacteria: Enterococcus faecalis,

Bacillus subtilis;
Antibacterial activity [5,7]
- Inhibiting activity against Gram-negative bacteria: Salmonella
typhimurium, Shigella sonnei, Helicobacter pylori.

Antifungal activity - Inhibiting activity against Candida glabrata strains. [7]

Antiviral activity - Inhibition of replication of Herpes simplex. [57]

Anti-allergic activity - Inhibition of histamine secretion in peritoneal cells of rats. [1,59]

- Normalization of alanine aminotransferase (ALAT), alkaline

phosphatase (ALP) and bilirubin;
Hepatoregenerative activity [10]
- Reduction in malonic dialdehyde (MDA), lipid peroxidation,
superoxide dismutase (SOD) and catalase.

- Inhibition of gluconeogenesis (naringenin, hesperitin);

- Inhibition of gluconeogenesis (naringenin, hesperitin);
Prevention of diabetes and
- Reducing wound-healing time; [1,60]
treatment of its symptoms
- Increasing tissue growth rate, collagen synthesis, and protein and
hydroxyproline concentration.

- Lowering blood lipids;

Anti-obesity activity [1,62]
- Reducing the levels of insulin, leptin and adiponectin in the blood.

- Limiting myocardial damage (naringenin);

Effects on the cardiovascular
- Decreasing blood fibrinogen; [1,11,63]
system
- Lowering blood pressure in people with hypertension.

Effects on the nervous system - Strengthening short-term memory. [67]

Effects on the respiratory system - Treatment of chronic pneumonia (naringenin). [68]

- Increasing bone density;

- Decreasing osteoclast activity;
- Decreasing TRAP-positive multinucleated cell numbers (nomilin);
Effects on the skeletal system [66,69]
- Decreasing bone resorption activity (nomilin);
- Down regulation osteoclast-specific genes (NFATc1 and TRAP mRNA
levels) (nomilin).

Treatment of menstrual
- Period induction in cases of irregular menstrual cycles. [5]
disorders
Plants 2020, 9, 119 16 of 24

Table 8. Biological activity of C. limon essential oil confirmed by scientific research.

Activity Mechanism of Action References

Anticancer activity - Stimulation of apoptosis of colorectal cancer cells. [70]

- Inhibiting cell migration;

- Inhibition of cytokine production;
- Inhibition of inflammation mediator (D-limonene);
Anti-inflammatory activity [49,52]
- Inhibition of leukocyte chemotaxis (D-limonene);
- Interaction with 5-lipoxygenase, TNF-α (tumour necrosis factor), IL-6
(interleukin-6).

- Inhibiting activity against Gram-positive bacteria: Staphylococcus

capitis, Micrococcus luteus, Bacillus subtilis;
Antibacterial activity [21,52,53]
- Inhibiting activity against Gram-negative bacteria: Pseudomonas
fluorescens, Escherichia coli.

- Inhibiting activity against: Aspergillus niger, Saccharomyces cerevisiae,

Antifungal activity [21,52,56]
Candida parapsilosis strains (D-limonene, β-pinene, citral).

Antiviral activity - Inhibition of the virus Herpes simplex. [57]

- Treatment of schistosomiasis caused by Schistosoma mansoni

Antiparasitic activity (D-limonene); [58]
- Inhibitory effect on Sarcoptes scabiei development.

- Inhibiting growth of Streptococcus mutans and its adhesion to enamel;

Anticaries activity [54]
- Inhibition of glucosyltransferase transcription and enzymatic activity.

- Stimulation of liver detoxification by activation of cytochrome P450

Hepatoprotective and
and liver enzymes (glutathione S-transferase) in chronic [71]
detoxification activity
liver poisoning.

- Decreased glycolized haemoglobin (D-limonene);

- Decreased gluconeogenesis enzymes: glucose-6-phosphatase and
Diabetes prevention [61]
fructose-1,6-biphosphatase (D-limonene);
- Decreased blood glucose (D-limonene).

- Lowering cholesterol and preventing fat deposits (D-limonene);

Anti-obesity activity - Equalization of blood sugar (D-limonene); [72]
- Regeneration of pathological changes in the liver and pancreas.

- Increased gastric motility and reduction of nausea (D-limonene);

- Neutralization of stomach acids (D-limonene);
Effect on the digestive system [21]
- Relief of gastric reflux (D-limonene);
- Increased bile flow.

- Reducing the level of triglycerides, LDL and increasing the level of

HDL cholesterol in the blood;
Lipolytic and
- Lowering cholesterol and arachidonic acid levels by stimulating liver [1,21,72]
cholesterol-lowering activity
enzymes and cytochromes;
- Lipolytic effect (γ-terpinene and p-cymene).

- Inhibitory effect on pain receptors similar to that of indomethacin and

hyoscine (D-limonene);
Effects on the nervous system [73]
- Sedative and anxiolytic effect by activating serotonin and
dopamine receptors.

8. C. limon in the Food Industry

Due to the rich chemical composition of C. limon fruit and other lemon-derived raw materials, they
have applications in the food industry and in food processing. The lemon fruit is used mainly as a fresh
fruit, but it is also processed to make juices, jams, jellies, molasses, etc. [41]. Fresh lemon fruit can be
Plants 2020, 9, 119 17 of 24

kept for several months, maintaining their levels of juice, vitamins, minerals, fibre, and carbohydrates.
The vitamin C (ascorbic acid) content in lemon fruits and juices decreases during storage and industrial
processing. The factors lowering this content are: oxygen, heat, light, time, storage temperature and
storage duration. To prevent the reduction in the ascorbic acid levels and antioxidant capacity of both
the lemon fruit and lemon juice, they should be kept at 0–5 ◦ C and protected from water loss by proper
packaging, with high relative humidity during distribution. Under such conditions, lemon products
show a good retention of vitamin C and antioxidant capacity [41,74].
C. limon peel is rich in pectin, which is used in a wide range of food industrial processes as a
gelling agent, including the production of jams and jellies, and as thickener, texturizer, emulsifier and
stabilizer in dairy products. Due to its jellifying properties, the pectin is also used in pharmaceutical,
dental and cosmetic formulations [75].
Lemon juice is used as an ingredient in beverages, particularly lemonade and soft drinks, and in
other foods, such as salad dressings, sauces, and baked products. Lemon juice is a natural flavouring
and preservative, and it is also used to add an acidic, or sour, taste to foods and soft drinks [41,76].
C. limon is the most suitable, being free from pesticide residues, raw material for enhancing the
flavour of liqueurs, e.g., “limoncello”, the traditional liqueur of Sicily. It is made by the maceration of
lemon peel in ethanol, water and sugar [41,76].
Currently, the essential oil from lemon, i.e., pure isolated linalol and citral, are used mainly as
a flavouring and natural preservative due to their functional properties (antimicrobial, antifungal,
etc.) [52,53]. In particular, they are often used to extend the short shelf-life of seafood products and in the
production of some types of cheese because they significantly reduces populations of microorganisms,
especially those from the family Enterobacteriaceae [41,76].

9. Cosmetological Applications
C. limon fruit extracts and essential oil, as well as the active compounds isolated from these raw
materials, have become the object of numerous scientific studies aimed at proving the possibility of
their use in cosmetology. Lemon-derived products have long been credited with having a positive
effect on acne-prone skin that is easily affected by sunburn or mycosis. In this regard, traditional uses
of this raw materials are known in various parts of the world. In Tanzania, the fruit juice of C. limon is
mixed with egg albumin, honey and cucumber, and applied to the skin every day at night to smooth
the facial skin and treat acne [77]. Juice from freshly squeezed fruit of C. limon mixed with olive oil is
used as a natural remedy for the treatment of hair and scalp disorders in the West Bank in Palestine [78].
Currently, knowledge of the cosmetic activity of C. limon is constantly expanding.
C. limon essential oil shows antibiotic and flavouring properties, and for this reason it is used in
formulations of shampoos, toothpaste, disinfectants, topical ointments and other cosmetics [41].
Scientific studies have shown a significant antioxidant effect of C. limon fruit extracts, which is the
reason they are recommended for use in anti-ageing cosmetics [8,48]. The use of different carriers for
C. limon extracts (e.g., hyalurosomes, glycerosomes) in cosmetics production technology contributes to
an even greater inhibition of oxidative stress in skin-building structures, including keratinocytes and
fibroblasts (Table 9) [79]. In addition, vitamin C from C. limon is used as an ingredient in specialized
dermocosmetics. Its external use increases collagen production, which makes the skin smoother
and more tense. It is used in anti-aging products, to reduce shallow wrinkles, and as a synergistic
antioxidant in combination with vitamin E [48].
The ingredients of C. limon essential oil (including citral, β-pinene, D-limonene), due to the
inhibiting activity of tyrosinase and the inhibition of L-dihydroxyphenylalanine (L-DOPA) oxidation,
have a depigmenting effect [80]. In addition, the essential oil has been proven to support the penetration
of lipids as well as water-soluble vitamins. It can be used as a promoter of penetration of active
substances through the skin [81]. Moreover, besides the direct effect on the skin, the essential oil can
also be used as a natural preservative and as a corrigent in cosmetic products. Studies have confirmed
its antibacterial and fungistatic effects (Table 9) [7,52,53].
Plants 2020, 9, 119 18 of 24

Furthermore, C. limon pericarp extracts, too, exhibit scientifically proven activity that helps to
accelerate the regeneration of diabetic wounds. In addition, the essential oil derived from C. limon
pericarp has shown anti-inflammatory, anti-allergic and slimming properties [49,59,60,62].

Table 9. Biological activity of C. limon fruit extracts, essential oil and its ingredients compounds
significant from the cosmetics point of view, confirmed by scientific research.

Extracts and
Activity Mechanism of Action References
Compounds Tested
Strong lipid peroxidation reduction and free radical
C. limon essential oil
reduction effect in vitro and in vivo. [79,80]
Antioxidant
activity Extract enclosed in hyalurosomes and glycerosomes
C. limon var. pompia fruit
reduces oxidative stress caused by hydrogen peroxide
extracts
and the viability of keratinocytes and fibroblasts.
Essential oil ingredients Essential oil components show tyrosinase inhibitory
Depigmenting
(e.g., citral, β-pinene, activity. Mixture of essential oil ingredients has a [80]
activity
D-limonene) stronger inhibitory effect due to their synergistic effect.
Acc. to in vitro study on human epidermal cells
Effect of (SkinEthic), C. limon essential oil increased the
increasing the penetration of α-tocopherol. Modification of TEWL
C. limon essential oil [81]
penetration of (Trans Epidermal Water Loss) was transient. C. limon
substances essential oil enhanced the penetration of locally
administered lipids and water-soluble vitamins.
Preservative Antibacterial activity and increasing the fungistatic effect
C. limon essential oil [7,52,53]
effect in cosmetics of synthetic preservatives.

According to the CosIng Database (Cosmetic Ingredient Database), C. limon can be used in
twenty-three forms. It can be used in cosmetics in the form of oils obtained from various organs, in
the form of extracts, hydrolates, powdered parts of the plant, wax and juice [27]. The most common
activity defined by CosIng for the raw material of this species is to keep the skin in good condition,
to improve the odour of cosmetic products, and to mask the smell of other ingredients of cosmetic
preparations [27]. The approved forms of raw materials and their potential effects, as well as their use
as corrigents, presented in the CosIng Database, are summarized in Table 10 [27].

Table 10. C. limon in cosmetic products according to CosIng.

The Form Activity

C. limon (lemon)/Fucus serratus extract skin conditioning
C. limon bud extract humectant, skin conditioning
C. limon flower water humectant, skin conditioning
C. limon flower/leaf/stem extract masking, skin conditioning, tonic
C. limon flower/leaf/stem oil masking
C. limon fruit extract masking, skin conditioning
C. limon fruit oil astringent, tonic
C. limon fruit powder skin conditioning
C. limon fruit water masking, skin conditioning
C. limon juice skin conditioning, tonic
C. limon juice extract tonic
C. limon juice powder skin conditioning, tonic
C. limon leaf extract perfuming
C. limon leaf oil perfuming, masking
C. limon leaf/peel/stem oil skin conditioning
C. limon peel masking, skin conditioning
C. limon peel cera/C. limon peel wax skin conditioning
C. limon peel extract emollient, skin conditioning, skin protecting, tonic
C. limon peel oil masking, perfuming, skin conditioning
C. limon peel powder absorbent, viscosity controlling
C. limon peel water skin conditioning
C. limon seed oil masking, perfuming, skin conditioning
Plants 2020, 9, 119 19 of 24

C. limon essential oil has been used since the 18th century in the production of the famous ‘Eau
de Cologne’. In aromatherapy, it is used to treat numerous diseases and lifestyle-related ailments:
hypertension, neurosis, anxiety, varicose veins, arthritis, rheumatism and mental heaviness. It also
alleviates symptoms characteristic of menopause. C. limon essential oil is also used in aromatherapy
massages to relax muscles, and for calming down and deep relaxation [21].
C. limon fruit extracts and essential oil should not be used in high concentrations in baths or
directly on the skin. Recent studies have shown that D-limonene contained in the oil has an allergenic
and irritating effect on the skin. It may cause cross-allergy with balsam of Peru. After applying
cosmetics containing C. limon oil, it is forbidden to expose the skin to sunlight. C. limon essential
oil contains photosensitizing compounds belonging to the linear furanocoumarin group. The lemon
pericarp contains: bergapten, phellopterin, 5- and 8-geranoxypsoralen, and the essential oil contains:
bergapten, imperatorin, isopimpinellin, xanthotoxin, oxypeucedanin and psoralen [21,82].
The International Fragrance Association (IFRA) has issued restrictions on the use of C. limon
essential oil. In preparations remaining on the skin, the concentration of that oil should not exceed 2%.
In addition, C. limon essential oil should not be used in preparations remaining on skin exposed to UV
rays. They should not contain more than 15 ppm of bergapten [83].

10. Plant Biotechnological Studies on C. limon

Plant biotechnology creates opportunities for the potential use of plant in vitro cultures in
the pharmaceutical, cosmetics and food industries. In vitro cultures can be a good alternative to
plants growing in vivo. Plant biotechnology enables control and optimization of the conditions for
conducting in vitro cultures to increase the accumulation of active compounds. It facilitates, among
other things, optimization of the culture medium, including the concentration of plant growth and
development regulators, the use of elicitors (stressors), the selection of highly productive cell lines and
genetic transformations. In vitro cultures can also be used in plant propagation (micro-propagation
process) [84].
C. limon cultures in vitro have thus far been the subject of research concerned with the development
of micropropagation protocols. They have focused on the selection of plant growth regulators (PGRs)
that induced shoot and root production in in vitro cultures. In 2012, biotechnological research on the
micropropagation of C. limon was performed by Goswami et al. [85] from SKN Rajasthan Agricultural
University in India. Shoot cultures were propagated from plant nodes on a Murashige and Skoog
(MS) medium [86] containing different types and concentrations of PGRs. The maximum number of
shoots and shoot regenerations was observed at a low level of 6-benzyladenine (BA) −0.1 mg/L, or
kinetin −0.5 mg/L. Shoot proliferiation was also observed in combinations of PGRs such as BA and
1-naphthaleneacetic acid in concentrations of 0.1 mg/L each. With an increase in BA concentration in
MS medium, shoot proliferation decreased. Regenerated shoots showed root induction on MS basal
medium or on MS medium containing 1.0 mg/L of indole-3-butyric acid.
Another biotechnological study on C. limon was carried out in the Department of Citriculture in
Murcia (Spain) [87]. The researchers studied organogenesis and made histological characterization of
mature nodal explants of two important cultivars of C. limon—‘Verna 51’ and ‘Fino 49’. The highest
number of buds per regenerating explant was obtained on the MS medium in comparison with the
Woody plant medium [88]. The presence of 1–3 mg/L BA, in combination with 1 mg/L of 1-gibberellic
acid (GA) in the culture medium, was essential for the development of adventitious buds. The lowest
extent of organogenesis was observed when BA was used in the medium without GA [87].

11. Conclusions
The presented review proves that C. limon is a very attractive object of different scientific studies.
The C. limon fruit is a raw material that can be used in different forms, e.g., extracts, juice and essential
oil. The rich chemical composition of this species determines a wide range of its biological activity and
its being recommended for use in phytopharmacology. The studies have focused on the essential oil
Plants 2020, 9, 119 20 of 24

and its main active compound—D-limonene. Extracts from C. limon fruits are rich in flavonoids such
as naringenin and hesperetin.
Current pharmacological studies have confirmed the health-promoting activities of C. limon,
especially its anti-cancer and antioxidant properties. C. limon also finds increasing application in
cosmetology and food production.
There has been some biotechnological research aimed at developing effective in vitro
micropropagation protocols for C. limon.

Author Contributions: Conceptualization, A.S., H.E. and M.K.-S.; data curation, A.S. and M.K.-S.;
writing—original draft preparation, A.S. and M.K.-S.; writing—review and editing, A.S., M.K.-S. and H.E.;
supervision, A.S. and H.E. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflicts of interest.

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