Antimicrobial and antioxidative activity of extracts and essential oils of Myrtus

communis L. _
Verica Aleksic, Petar Knezevic∗

Abstract: Since synthetic antimicrobial agents and food additives can cause a number of adverse
effects, there is a growing interest from consumers in ingredients from natural sources. Medicinal
plants, such as Myrtus communis L. are a source of new compounds which can be used in both the
food industry and for medical purposes, primarily as antimicrobial agents. In this review, the
characteristics of myrtle essential oils and extracts are summarized, with particular attention to their
chemical composition, biological activities and potential applications.

1. Introduction

Myrtle (Myrtus communis L.) is a medicinal plant endemic to the Mediterranean area and it has
been used by locals for its culinary and medicinal properties since antiquity (Atzei 2003). This is a
well-established tradition in many countries however, despite the increasing scientific interest in
this field, there is a lack of summarized data on herbal medicine composition, therapeutically
applications and risks connected to their consumption. Therefore, this review summarizes results
regarding chemical composition and biological activities of M. communis L.

1.1. Myrtus communis L.

Common myrtle belongs to the Myrtaceae family, which comprises approx. 145 genera and over
5500 species (Snow et al. 2011).The genus Myrtus includes flowering plant with approximately 16
species reported in areas of the Middle East and Asia (Twaij et al.1988; Romani et al. 1999). M.
communis L., known as true myrtle, is one of the important aromatic and medicinal species from
this family. It is an evergreen sclerophyll shrub or small tree, 1.8–2.4 min height, with small foliage
and deep fissured bark (Mendes et al.2001). True Myrtle is characterized by its branches, which
form a close full head, thickly covered with ovate or lanceolate ever green leaves (Fig. 1). Their
leaves are 3–5 cm long and contain tannins, flavonoids and volatile oils (Baytop 1999). This species
is a very aromatic plant because of the high essential oil content in its leaf, flower and fruit glands.
It has solitary axillary white or rosy flowers, followed by black a several-seeded berry which is
spherical in shape with dark red to violet in color (Mahmoud et al. 2010). There are two major fruit
morphologies based on the color whether dark or white. The dark color is more frequent, but there
are also cultivated white-colored types, which yield much larger fruits than their wild counterparts
(Klein et al. 2000).

1.2. Distribution Myrtle (M. communis L.) is a common part of typical Mediterranean flora.

The plant grows abundantly from the north western to the eastern Mediterranean, including
bordering countries and western Asia, as well as Aegean regions (Baytop 1997). Myrtle is native to
southern Europe, North Africa and west Asia. It is also distributed in Southern America,
northwestern Himalaya and Fig. 1. Myrtus communis L. plant (by courtesy of Prof. Michael
Pascoe,www.wordplants.ca).Australia. Myrtle is cultivated in gardens, especially in Northwest
Indian region, because of its fragrant flowers (Nadkarni 1989).Being widespread throughout the
Mediterranean region, the species is one of the most important evergreen shrubs in the
Mediterranean maquis. In Italy it grows along the coasts and on the internal hills and it is abundant
especially on the islands, where it represents one of the most characteristic species (Cannas et
al.2013). In Portugal, myrtle grows wild mainly in the central and southern parts of the country. The
genus Myrtus, in Tunisia, is rep-resented by only one species, M. communis L., which grows wild
in the coastal areas, the internal hills, and the forest areas of northern Tunisia. Two myrtle varieties
are described in old local Tunisian flora: M. communis var. italica L. and M. communis var. baetica
L.(Pottier-Alapetite et al. 1979), which possesses the same vegetative characters. The
morphological difference between the two varieties regards to size of fruits and leaves. This herb
grows spontaneously Iran, Spain, France, Greece, Turkey, Algeria, Morocco, Croatia and
Montenegro (Naserian 1997; Chryssavgi et al. 2008; Mimica-Duki´cet al. 2010; Berka-Zougali et
al. 2012; Mahmoud et al. 2010; Jerkovicet al. 2002; Gauthier et al. 1988).1.3. Traditional
application Myrtle has been used since ancient times as a spice, as well as for medicinal and food
preparation purposes. Myrtle as a spice finds no wide application because of its bit-terness, despite
the pleasant odor. The taste is very intense, quite unpleasant and strongly bitter, so its culinary
application is limited to the region of origin, such as Italy (Gortzi et al. 2008). In Italy, especially in
Sardinia, berries and leaves are used to produce two well-known liquors (Mirto Rosso and Mirto
Bianco, respectively)(Messaoud et al. 2012). Foods flavored with the smoke of myrtle are common
in rural areas of Italy or Sardinia (Gortzi et al. 2008).However, some parts of the plant are used in
the food industry, for flavoring meat and sauces (Chalchat et al. 1998), and its berries and leaves are
mostly employed for the industrial formulation of sweet liquors with advertised digestive properties
(Clark 1996; Mulaset al. 2000).Its leaves are very fragrant and have been extensively used in the
perfume and cosmetic industries, particularly in Portugal (Clark1996) as well as Turkey (Baytop
1999).It is traditionally used as an antiseptic, disinfectant and hypo-glycemic agent (Elfellah et al.
1984). In Turkey myrtle leaves as well as fruits have been used as an antiseptic medicine in villages
(Baytop 1999). Similarly, in Italian folk medicine, the fruit of this plant is used in the treatment of
many types of infectious disease, including diarrhea and dysentery; the leaves are used as antiseptic
and antiinflammatory agent, as well as a mouthwash, for the treatment of candidiasis (Gortzi et al.
2008). The essential oil obtained from myrtle leaves has been used in the treatment of lung
disorders (Clark 1996). In traditional medicine, myrtle is frequently consumed as an infusion and
decoction (Le Floch 1983). Generally, in folk medicine, a decoction of leaves and fruits is used
orally for the treatment of stomach aches, hypoglycaemia, disbiosis, cough, constipation, poor
appetite, as well as also externally for wound healing (Serce et al. 2010).Different parts of the
myrtle plant traditionally have assorted specific applications (Table 1). Infusions made from the
leaves and young branches are approved to be stimulant, antiseptic, astringent and hypoglycemic,
and they are considered to be a health remedy for asthma, eczema, psoriasis, diarrhea,
gastrointestinal disorders and urinary infections (Ziyyat et al. 1997). The leaf decoction is used for
vaginal washing, enemas and against respiratory diseases (Marchini and Maccioni 1998), while
decoction from the fruits is used as antidiarrheal, antihemorrhoidal agents and in mouth and eyes
disease treatment (Ziyyat et al. 1997). Flowers are traditionally used against varicose veins, and for
preparing capillary lotions (LeFloch 1983).Other uses of its leaves include cattle feed, cut foliage
and potted plants (Bruna et al. 2007).

2. Extraction procedures
Extraction techniques separate the soluble plant metabolites through selective use of solvents. The
process of obtaining plant extracts consists of several steps – collection and authentication of plant
material, drying, size reduction, extraction, filtration, concentration, and final steps are further
drying and reconstitution (Handaet al. 2008). It is important to emphasize that the quality of an
extract is influenced by several factors such as the plant parts used as starting material, the solvent
used for extraction, the extraction procedure, and the solvent ratio. The solvents most commonly
used for gaining extracts are water, methanol, ethanol and ethylacetate (Chryssavgi et al. 2008;
Amensour et al. 2010; Tuberoso et al. 2010).Conventional methods used in extraction procedures
from plants include soxhlet extraction, thermal desorption, maceration, phytonic desorption,
infusion, extraction leaching, surfactant mediated extraction, accelerated solvent extraction,
pressurized liquid extraction, steam distillation, percolation, membrane process, decoction, sample
disruption method, counter current extraction and enfleurage (Handa et al. 2008).According to
European Pharmacopeia (2002) for essential oils preparation, it is needed air dried plant materials
such as leaves, flowers, fruit, berries and branches. The oils contained within plant cells are
liberated through heat and pressure from these parts of the plant matter, and the color may vary
from a pale to deep yellow depending of the plant part used. The extraction of essential oils from
plant material can be achieved by various methods, of which the most commonly used methods
include hydro-distillation(with a collecting solvent which is then removed under vacuum),steam and
steam/water distillation (Fig. 2) (Bowles 2003; Margariset al. 1982; Surburg and Panten 2006). In
addition to these conventional methods, other newer methods for extracting essential oils use
refrigerant hydrofluorocarbon solvents at low temperatures, resulting in good quality of the
extracted oils as well as sol-vent extraction, effleurage, aqueous infusion, cold or hot pressing,
supercritical fluid extraction, solvent free microwave extraction(SFME) and phytonic process (Da
Porto et al. 2009; Hunter 2009;Lahlou 2004; Martínez 2008; Ferhat et al. 2006; Pourmortazavi and
Hajimirsadeghi 2007; Surburg and Panten 2006). Berka-Zougaliet al. (2012) showed that SFME is
highly effective for reducing extraction time (30 min for SFME against 180 min for hydro-
distillation), providing an essential oil with a chemical composition enriched in oxygenated
compounds and has essential oils yields similar to those when hydro-distillation method applied.
Also, it had been found that hydro-distillation and steam-distillation methods yield oils rich in
terpene hydrocarbons, while in contrast, the supercritical extracted oils contained a higher
percentage of oxygenated compounds (Donelian et al. 2009; Eikani et al. 2007;Reverchon 1997;
Wenqiang et al. 2007). This implies that the quantitative and qualitative chemical composition of
the essential oil differs according to the applied extraction technique. Thus, after the essential oil
extraction, it is necessary to determine its qualitative and quantitative characteristics.
Quantities/yields of the essential oils are usually determined gravimetrically, while qualitative
analysis of the essential oils is usually performed by gas chromatography (GC) or gas
chromatography–mass spectrometry (GC–MS) method (Mazza, 1983).3. Chemical composition of
Myrtus communis L. extractsand essential oils M. communis L. main secondary metabolites are
polyphenols and essential oils. Myrtus species have been reported as very rich involatile oils
(Satrani et al. 2006; Shikhiev et al. 1978; Tuberoso et al.2006), phenolic acids (Romani et al. 1999),
flavonoids (Romaniet al. 1999; Joseph et al. 1987), tannins (Diaz and Abeger 1986), anthocyanin
pigments (Martin et al. 1990) and fatty acids (Cakir2004). Previous studies on M. communis L.
aerial parts have also revealed the presence of several specific chemical compounds. For example,
the dried leaves of this herb contain 1,8-cineole(13.5–19.6%), linalool (7.7–15.8%), linalyl acetate
(2.5–6%),terpineole, terpinolene, tannins and flavonoid compounds(Chryssavgi et al. 2008). Leaf
and flowers contain essential oils, phenolic acids, flavonoids and tannins (Messaoud et al.2005;
Aidi Wannes et al. 2010). Berries are composed of tannins, anthocyanins (0.2–54%), fatty and
organic acids (9–52%), and its content depends on used extraction solvent and/or ripening
period(Martin et al. 1990; Tuberoso et al. 2010; Messaoud et al. 2012).It is evident that the content
of these compounds also differs depending on the plant part used (Tables 2 and 3), but generally the
most common compounds found in myrtle leaves, steams and flowers are pinene (∼10–60%) and
1,8-cineole (∼12–34%) (AidiWannes et al. 2010).

3.1. Myrtus communis L. essential oil compositions

Essential oils are odorous and volatile compounds found only in 10% of the plant kingdom (Djilani
and Dicko 2012). Essential oils and their components can be very promising biological agents,
because of their relative safety, wide acceptance by consumers and exploitation for potential
multipurpose use (Ormancey et al. 2001;Sawamura 2000). They are stored in plants in special
brittle secretory structures, such as glands, secretory hairs, secretory ducts, secretory cavities or
resin ducts (Ahmadi et al. 2002; Bezic et al.2009; Ciccarelli et al. 2008; Gershenzon 1994; Liolios
et al. 2010;Morone-Fortunato et al. 2010; Sangwan et al. 2001; Wagner 1996).The total essential oil
content of plants is generally very low and rarely exceeds 1% by mass (Bowles 2003). For example,
the essential oil yields in leaf, stem and flower of M. communis var. italic L. were respectively
0.61%, 0.08% and 0.30% (w/w) (Aidi Wanneset al. 2010). Essential oils are hydrophobic and thus
only slightly soluble in water. They are soluble in alcohol, non polar or weak lypolar solvents,
waxes and oils. Most essential oils are colorless orpale yellow, liquid and have lower density than
water (Gupta et al.2010; Martin et al. 2010). Essential oils are complex mixtures comprising many
various compounds. Chemically they are derived from terpenes and their oxygenated compounds
(Prabuseenivasan et al.2006).The chemical composition of the myrtle essential oil has been
described by many authors (Boelens and Jimenez 1991,1992; Bradesi et al. 1997; Chalchat et al.
1998; Ozek et al.2000; Koukos et al. 2001; Aidi Wannes et al. 2007, 2009;Mimica-Duki´c et al.
2010; Messaoud et al. 2012; Kafkas et al.2012). Compounds that have been found in myrtle oils
includeE-2-hexenal, Z-3-hexenol, hexanol, tricyclene, _-thujene, _-pinene, sabinene, _-pinene,
myrcene, _-3-carene, _-terpinene,p-cymene, limonene, 1,8-cineole, E- _-ocimene, linalool E-
oxide, terpinolene, linalool, terpinene-4-ol, borneol, p-cymene-8-ol, _-terpineol, myrtenol, nerol,
cis-carveol, geraniol, linalyl acetate, bornyl acetate, eugenol, myrtenyl acetate, terpinyl acetate,
geranyl acetate, neryl acetate, methyl eugenol, caryophyllene, humulene, alloaromadendrene,
germacrene-D, thiophene, ger-anyl 2-methylbutyrate, spathulenol, nonadecane, elemene,
caryophyllene oxide, camphene, phellandrene, erpinene, cis-linalool oxide, trans-linalool oxide
and tridecane. All these M. communis L. essential oil compounds maybe classified into three main
categories: terpenes (monoter-pene hydrocarbons and sesquiterpene hydrocarbons),
terpenoids(oxygenated monoterpenes and oxygenated sesquiterpenes) andphenylpropanoids
(Andrade et al. 2011; De Sousa 2011; Griffinet al. 1999; Lis-Balchin 1997; Sangwan et al. 2001),
but also intohydrocarbons and oxygenated compounds (Akhila 2006; Halm2008; Hunter 2009;
Margaris et al. 1982; Pourmortazavi and Hajimirsadeghi 2007; Shibamoto et al. 2010).Terpenes
form structurally and functionally different classes. Terpenes are hydrocarbons produced from
combination of several five carbon base units called isoprene units (C5H8). They are synthesized in
the cytoplasm of plant cells, and the synthesis proceeds via the mevalonic acid pathway starting
from acetyl-CoA. Terpenes have a hydrocarbon backbone which can be rearranged into cyclic
structures by cyclases, thus forming monocyclic or bicyclic structures (Caballero et al. 2003). The
main terpenes are monoterpenes (C10H16) and sesquiterpene (C15H24), but longer chains also
exist. Examples of terpenes found in myrtle essentialoils include pinene, limonene, sabinene and
myrcene. Terpenoids are terpenes that undergo biochemical modifications via enzymesthat add
oxygen molecules and move or remove methyl groups (Caballero et al. 2003). Terpenoids can be
sub-divided into alcohols, esters, aldehydes, ketones, ethers, phenols and epoxides. Exam-ples of
myrtle terpenoids are myrtenol, linalool, linalyl acetate and geraniol (Table 2). The main terpenes in
myrtle essential oils are the monoterpenes and sesquiterpenes and its terpenoids (Aidi Wanneset al.
2009, 2010; Deriu et al. 2007; Mimica-Duki´c et al. 2010). The monoterpenes formed from the
coupling of two isoprene units are the most representative molecules constituting 90% of the
essential oils which allow a great variety of structures. Various groups of organic compounds called
phenylpropanoids are synthesized from the aminoacid precursor phenylalanine in plants.
Phenylpropanoids have their name from the six-carbon aromatic phenol group and the three-carbon
propene tail of cinnamic acid, produced in the first step of phenylpropanoid biosynthesis.
Phenylpropenes constitute a relatively small part of myrtle essential oils, and those that have been
most thoroughly studied are eugenol, isoeugenol, vanillin, safrole and cinnamaldehyde (Hyldgaard
et al. 2012).Environmental factors were considered to play a key role in the chemical composition
of myrtle oil (Scora 1973). The fragrance and chemical composition of essential oils can vary
depending on the geoclimatic location and growing conditions, including concentration of nutrients,
temperature, humidity, soil type, day length, climate, altitude, amount of available water, ect. The
chemical com-position also depends on season or vegetative period of plant, i.e. before or after
flowering (Andrade et al. 2011; Deans et al. 1992; Margaris et al. 1982; Pengelly 2004; Sangwan et
al. 2001). According to these factors, plant biosynthetic pathways can change the relative proportion
of the primary oil components. These variations in chemical composition led to the notion of
chemotypes, which are generally defined as a distinct population within the same species that
produces different chemical profiles for a particular class of secondary metabolites (Djilani and
Dicko 2012). There are different essential oil chemotypes which can distinguish myrtle oil of
different origins, as well as seasonal variations throughout the vegetative cycle of plants (Bradesi et
al. 1997; Chalchat et al. 1998; Flaminiet al. 2004; Chryssavgi et al. 2008). Bradesi et al. (1997)
proposed classification of M. communis L. essential oil on the basis of myrtenylacetate content, in
two chemotypes. Each chemoptype group can be further divided into two subgroups, according to
the relative ration of pinene to myrtenyl acetate or pinene to cineole. In addition, plant genotype is
another important factor that influences the chemical composition of essential oils (Djilani and
Dicko 2012).Therefore, all these biotope factors, genetic and epigenetic, influence the biochemical
synthesis of essential oils in a particular plant. Thus, the same species of plant can produce a similar
essential oil, but with different chemical composition and therapeutic activities. Essential oil
composition also depends on the plant parts used for oil preparation. Chryssavgi et al. (2008)
determined that the Myrtus caommunis essential oil from leaves contain 42 com-pounds, while
according to Aidi Wannes et al. (2010) there are 44 components constituting Myrtus communis
essential oil from leaves, stems and flowers. The same group of authors examined myrtle fruit
essential oil composition during its ripening and identified 47 compounds whose concentration
fluctuated during its different stages of ripening (Aidi Wannes et al. 2009).Although monoterpenes
are dominant in essential oils (70–90%) the distribution of oxygenated monoterpenes and
monoterpene hydro-carbons vary. Aidi Wannes et al. (2010) showed that monoterpene
hydrocarbons dominate in leafs, followed by oxygenated monoterpenes. In stems dominated
oxygenated monoterpenes, followed by monoterpenes hydrocarbons and sesquiterpene
hydrocarbons. According to them, the flower is characterized by high levels of monoterpene
hydrocarbons and oxygenated monoterpenes with an appreciable percentage of phenypropanoids.
The major constituents of the leaf essential oil composition are pinene, 1,8-cineole and _-pinene.
The main essential oil components in theflower are pinene and 1,8-cineole, as well as other
compounds including limonene, eugenol, _-terpineol, linalool and methyleugenol. 1,8-Cineole is
the dominant component in stem essential oil and it is followed by _-pinene, E- _-ocimene and
linalool(Aidi Wannes et al. 2010). Analysis of myrtle fruit essential oil composition showed that the
main monoterpene compounds are 1, 8-cineole, geranyl acetate, linalool and pinene (Aidi
Wanneset al. 2009).As shown, there is considerable variability in the composition of myrtle
essential oil depending on multiple biotope factors, but the most important constituents of myrtle oil
are terpenoids myrtenol, myrtenol acetate, limonene, linalool, _-pinene, 1,8-cineole, _-
caryophyllenein, as well as p-cymene, geraniol, nerol and the phenylpropanoid – methyleugenol
(Ozek et al. 2000; Flaminiet al. 2004; Deriu et al. 2007; Chryssavgi et al. 2008; Aidi Wanneset al.
2009, 2010; Mimica-Duki´c et al. 2010; Messaoud et al. 2012)(Table 2).3.2. Myrtus communis L.
extracts composition M. communis L. extracts profile constitutes polyphenolic com-pounds, which
are grouped in three major chemical classes –phenolic acids, tannins and flavonoids (Table 3).
Phenolic acids, components of M. communis L. extracts are garlic, ellagic, caffeic, syringic, vanillic
and ferulic acid. Tannins as another essentialoil component comprise hydrolysable tannins
(gallotannins) and proanthocianidins (condensed tannins). Flavonoids found in myrtle extracts are
myricetin, quercetin, catechin, and their derivates. Some of the myricetin and quercetin derivatives
(flavonols) foundin myrtle extracts are myricetin-3-d-galactoside, myricetin-3-d-rahmnoside,
quercetin-3-rutinoside, quercetin-3-d-rahmnoside, and catechin derivatives (flavanols) (Aidi
Wannes et al. 2010;Tuberoso et al. 2010; Montoro et al. 2006b; Romani et al. 1999).Extracts
composition may significantly vary, depending on plant organ used for extraction (Tuberoso et al.
2010; Aidi Wannes et al.2010; Hayder et al. 2008; Montoro et al. 2006a, 2006b; Piras et al.2009;
Romani et al. 2004). Amensour et al. (2009) showed that leaf extracts contain significantly higher
amount of total phenolic com-pounds than berry extracts. Yoshimura et al. (2008) identified ten
phenolic compounds from myrtle leaf including four hydrolysable tannins (oenothein B,
eugeniflorin D2, tellimagrandins I and tellimagrandins II), two related polyphenolic compounds
(gallic acidand quinic acid 3,5-di-O-gallate), and four flavonols (myricetin3-O- _-d-xyloside,
myricetin 3-O- _-d galactoside, myricetin 3-O- _-d-galactoside 6-O-gallate and myricetin 3-O- _-l-
rhamnoside).The contents of total phenols, tannins, flavonoids and proanthocyanidins vary among
myrtle parts. According to Aidi Wanneset al. (2010), leaf and flower are particularly rich in total
tannins. Since the proanthocyanidins are weakly presented, the authors suggested that leaf and
flower tannins belong to hydrolyzed tannin class. However, they estimated that the myrtle stem is
poor in tannins and moderately rich in flavonoids (catechin). Flavonols and flavanols in M.
communis L. leaves are detected in relatively large amounts (Aidi Wannes et al. 2010), with the
exception of quercetin derivatives and phenolic acids, which were found only in small amounts
(Romani et al. 1999, 2004).Except myrtle plant parts, finale extract composition depends also on
extraction solvent used for extract preparation, mainly because of its polarity (Lapornik et al.,
2005). The most commonly used solvents for myrtle extract preparation are water, alcohol
(methanol or ethanol), ethyl acetate, diethyl ether and chloroform. Tuberoso et al. (2010) proved
that ethanol and water extracts showed higher amount of extracted compounds in comparison to
ethyl acetate extracts, but the highest antiradical and antioxidant activities were found in ethanol
and ethyl acetate extracts. According to them ethanol extracts has the highest content of phenolic
compounds. In addition, their results showed a highly significant correlation between the amount of
total phenols and antiradical or antioxidant activities in myrtle leaf extracts. According to this it is
important to compare and explore the variance of phenol composition from various myrtle extracts,
since this heterogeneous repartition of bioactive substances entrained the variability of their
potential antimicrobial and antioxidant activities.

3.3. Activity of Myrtus communis L. compounds

Antimicrobial (antibacterial, antifungal and antiviral) and antioxidant properties of compounds

produced by M. communis L. have been reported in numerous studies (Pereira et al.2012; Othman
et al. 2007; Shan et al. 2007; Funatogawa et al.2004; Naserian 1997; Rattanachaikunsopon and
Phumkhachorn2010). For example, 1,8-cineole, linalool, eugenol, _-terpineol and _-terpinene, as
myrtle essential oils components have a good bactericidal effect against some Gram positive and
Gram negative bacteria (Oyedemi et al. 2009; Randrianarivelo et al. 2009).According to
Randrianarivelo et al. (2009) results, linalool MICs and MBCs values ranging 0.18–5.88 mg/ml and
0.18–11.75 mg/ml, respectively; while this values for 1,8-cineole ranged from 0.37 to11.75 mg/ml
and 0.73 to 11.75 mg/ml, respectively. Also, Zanettiet al. (2010) showed that some myrtle single
compounds such as limonene (0.17–2%), 1,8-cineole (2–16%) and _-pinene (1–16%)have
significant activity against M. tuberculosis strains. These bio-logical effects of myrtle essential oils
and extracts are result of activity of compounds belonging to various chemical classes. For example,
in the mid-1970s, scientists reported the isolation of a phloroglucinol antibiotic from the myrtle
leaves, named asmyrtu commulone A, and along with it they isolated more polar and abundant
phloroglucinol 6, named semi myrtucommulone(Appendino et al. 2002). It is proved that these
myrtle compounds possess significant activity against S. aureus strains, with MICs values in range
0.5–2 _g/ml for myrtucommulone A, and 32–64 _g/ml for semi myrtucommulone (Appendino et
al. 2002). These low MIC concentrations are the consequence of pure compounds application,
which in mixture with other compounds influence on enhancing of essential oils antimicrobial
activity. The complexity of extracts or essential oils chemical composition suggests involvement of
various action mechanisms and consequently, it is difficult to identify just one pathway of
molecular action. It is very likely that each of the constituents of the essential oils/extracts has its
own mechanism of action (Djilaniand Dicko 2012) or in the other hand, the compounds could acton
synergistic way. For instance, various synergistic antimicrobial activities have been reported for
constituents or fractions of essential oils when tested in binary or ternary combinations (Delaquiset
al. 2002; Pei et al. 2009; García-García et al. 2011; Nguefack et al.2012).

3. Antimicrobial assays for plant extracts and essential oils

Two methods for determining the antimicrobial properties of extracts and essential oils that are the
most widely used are agar diffusion method and broth dilution method. The water insolubility,
complexity and volatility, of extracts and especially essential oils are among their primary
characteristics and these properties influence the assessments of antimicrobial activities. The agar
dif-fusion method is not considered an ideal method for essential oils, as their volatile components
are likely to evaporate with the dispersing solvent during the incubation time, while their poorly
soluble components do not diffuse well in the agarised media. This problem can overcome by using
Tween 80 and DMSO (dimethylsulfoxide) to enhance the solubility of oils. Since the method is
relatively simple, it still remains the most commonly used technique (Prabuseenivasan et al. 2006;
Davidson and Parish 1989).The inhibitory effect of plant extracts and essential oils in the test tubes
and microtitar plates is measured turbid metrically or with the count plate method. The results are
usually presented as values of minimal inhibitor concentration (MIC) and minimal bactericidal
concentration (MBC) (CLSI 2006). In experiments testing the extracts and essential oils activity
toward microorganisms, results depends mainly on the method used, however a number of other
factors should also be considered. Culture conditions and initial number of bacterial cells
predominantly influence the analysis; therefore they should be precisely stated in reports (Kalemba
andKunicka 2003).

5. Biological effects of Myrtus communis L.

5.1. Antibacterial effect

The problems regarding application of conventional antibiotics, including antimicrobial resistance,

environmental problems, cancerogenity, side effects and high costs, have reinforced a tendency to
replace synthetic antimicrobials with natural alternative agents (Gortzi et al. 2006). Plant based
products are among the alternative agents examined in order to replace conventional antibiotics
(Harikrishnan et al. 2003; Immanuel et al. 2004; Shahidi-Bonjar2004a, 2004b). Accordingly,
extensive research has been carried out in order to evaluate the antimicrobial effect of the essential
oils and extracts which showed the ability to inhibit the growth of various pathogenic
microorganisms (Ayatollahi-Moosavi et al. 1996)(Table 4).The antibacterial properties of myrtle
essential oils and extracts against pathogenic bacteria were reported in many studies and obtained
results are promising (Table 4).Taheri et al. (2013) indicated antibacterial effect of myrtle leaves
hydroalcoholic extract on some pathogenic bacteria, particularly Staphylococcus aureus and Vibrio
chloreae. Mansouri et al. (2001) also proved antibacterial activity of several myrtle extracts and
essential oils and estimated that they have different activities because of its different constituting
compounds. Crude extracts of myrthle was tested against 6 Gram positive (S. aureus,
Micrococcusluteus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae,
Listeria monocytogenes) and 4 Gram negative bacteria (Escherichia coli, Proteus vulgaris,
Pseudomonas aeruginosa and Campylobacter jejuni), and inhibited the growth of all tested bacteria
except C. jejuni. MICs range from 0.1 for S. aureus and M. luteus to over 2 mg/ml for E. coli. The
group of authors made diethyl ether, ethyl acetate, and ethanol extracts. The diethyl ether extracted
fraction showed the highest level of activity with a MIC 0.025 mg/ml for S. aureus and M. luteus
and 0.1 mg/ml for E. coli and P. aeruginosa. Alem et al. (2008) observed similarity between MIC
and MBC for myrtle crude preparation, which vary from 0.5 ml/l for S. aureus to 45 mg/ml for
E.coli. It is also interesting to mention that the antibacterial activity of myrtle was markedly
increased by 18 times after it has been autoclaved at 121◦C for 15 min. When tested against 150
strains isolated from burns (predominantely P. aeruginosa i S. aureus), aqueous leaves extracts of
M. communis gave an excellent effect on bacterial growth and their effects were located within the
limits of antibiotic effects (Al-Saimary et al. 2002).High antibacterial activity of methanol, ethanol,
and ethylacetate leaf and berry mythle extracts was observed when it tested aginst food borne
pathogens. The methanolic leaf extract of M. communis showed antibacterial activity even against
L. monocytogenes and P. aeruginosa, but none of the extracts was active against E. coliK12
(Amensour et al. 2010). The activity of methanolic extract was also confirmed in other studies, such
as Gortzi et al. (2008). Hydroalcocholic extracts of myrthle leaves inhibited S. aureus with a low
MIC (0.2 mg/ml); it was less effective against E. coli and V. cholera (8 and 2 mg/ml, respectively)
and ineffective against P. aeruginosa (Taheri et al. 2013). In this study, detected MBC was 5–10
times greater than MIC. M. communis infusion, prepared from dry leaves, showed lower activity
against examined Gram negative bacteria, with MIC varied from 12.5 to 50 mg/ml (Messaoud et al.
2012).M. communis L. extracts profile constitutes polyphenolic compounds phenolic acids, tannins
and flavonoids, whose antimicrobial activity varies. Some results have indicated that phenolic
compounds significantly contributed to the antibacterial activity (Shan et al. 2007). This activity
may be attributed to the enzyme inhibition by the more oxidized phenolic compounds possibly
through reaction with sulfhydryl compounds or through more non-specific interactions with the
protein (Schelz et al. 2010). Cinnamic acid and caffeic acid were shown to be toxic to
microorganisms (Cowan 1999). Tannins as polymeric water-soluble phenols are commonly present
in higher herbaceous and woody plants. Tannins are reported to possess free radical scavenging
activity and also have antibacterial effects (Akiyama et al. 2001). Hydrolysable tannins have
antibacterial potential against Helicobacter pylori and it seems promising in the eradication of the
bacterium without affecting intestinal microbiota (Funatogawa et al. 2004). A great deal of
flavonoids is synthesized by plants to fight against bacterial infections, therefore it is no surprise
that they exhibit in vitro antimicrobial activity (Tsuchiya et al. 1996; Cushine and Lamb2005) and
the similar refers to catechins (Gradisar et al. 2007).Antibacterial activity of essential oils has been
demonstrated in many studies. In the study of Akin et al. (2010) the oils showed considerable
activity against most Gram negative and positive bacteria at concentration of 0.5% (v/v). It is
interesting to notice that oils were also effective in concentrations 0.01–2.5% against Helicobacter
pylori, which is a main cause of duodenal ulcer (Deriu et al.2007), while the extracts of Malpighia
emarginata fruit were effective against H. pylori with MICs values between 17 and 27
_l/ml(Motohashi et al. 2004). They also showed activity against food-borne pathogen Salmonella
Typhimurium when vegetables were washed with oils concentration of 500–1000 ppm (Gündüz et
al.2009). Against well known resistant bacteria M. tuberculosis myrtle essential oils showed activity
in concentration 0.17%, but not against M. avium subsp. para tuberculosis (>2%) (Zanetti et
al.2010). Similarily, low level of activity has been observed against P. aeruginosa, which is well
known bacterium notoriously resistant to antibiotics (Owlia et al. 2009).Randrianarivelo et al.
(2009) showed that major myrtle oil compounds oxygenated terpenes, such as 1,8-cineole, linalool
and terpineol, exhibit potent antibacterial activity. The antimicrobial activity of most terpenoids is
linked to their functional groups and it has been shown that the hydroxyl group of phenolic
terpenoids and the presence of delocalized electrons are important for antimicrobial activity. For
example, the antimicrobial activity of the carvacrol derivatives (carvacrol methyl ether and p-
cymene)were much lower than carvacrol (Dorman and Deans 2000; Ulteeet al. 2002; Ben Arfa et
al. 2006). Furthermore, it has been shown that phenylpropanoids antimicrobial activity depends on
the type and number of substituents on the aromatic ring, tested microbial strains, and various
experimental test parameters, including growth medium, temperature, etc. (Pauli and Kubeczka
2010).In vitro tests indicated that terpenes are inefficient as antimicrobials when applied as single
compounds (Dorman and Deans 2000;Koutsoudaki et al. 2005; Rao et al. 2010), while certain
terpenoid components of essential oils can act as un couplers, interfering with proton translocation
over a membrane vesicle and subsequently interrupting ADP phosphorylation (Schelz et al.
2010).Terpenoids may serve as an example of lipid soluble agents which affect the activities of
membrane-catalyzed enzymes exhibiting action on respiratory pathways. Specific terpenoids with
functional groups, e.g. phenolic alcohols or aldehydes, also interfere with membrane-integrated or
associated enzymes and/or proteins, stopping their production or activity (Kalemba and Kunicka
2003).It is interesting to mention that there is a report on application of liposomes and M.
communis extracts, with aim to overcome the problem of bacterial resistance (Gortzi et al. 2008).
Due to their unique properties, liposomes are able to enhance the effects of products by increasing
ingredient-solubility (easier incorporation of water-soluble compounds into oil-based products),
improving the bioavailability and the in vivo and in vitro stability (Gortzi et al.2006). The extract
from the herbal parts of M. communis L. possessed antimicrobial activity, which dramatically
increased after the encapsulation in liposomes. These findings indicate that encapsulated myrtle
extracts can be used as potent preservative, not only in food industry but also in cosmetics and
pharmacology. In order to control the growth of multi-drug resistant bacteria, essential oils could
also be combined with other antimicrobial agents, such as bacteriophages. Synergistic effect of
these two agents represents a potential solution to replace antibiotics. Indeed, it has been shown that
damage of bacterial cell membrane caused by essential oils, either facilitates penetration of
bacteriophages with subsequent replication inside the bacterial cell, or is supported by simultaneous
action of bacteriophages and essential oils on the cell membranes (Volodymyrivna Kon and ans
Kumar Rai 2012).

5.1.1. Mode of antibacterial action

Different modes of action are involved in the antimicrobial activity of essential oils and extracts.
Because of the variability of quantity and chemical profiles of the essential oil and extract
components, it is likely that their antimicrobial activity is not due to a single mechanism. It is
considered that these components have several sites of action at the cellular level. Generally, there
are six possible mechanisms of antimicrobial action, which include:(1) disintegration of
cytoplasmic membrane, (2) interaction with membrane proteins (ATPases and others), (3)
disturbance of the outer membrane of gram negative bacteria with the release of
lipopolysaccharides, (4) destabilization of the proton motive force with leakage of ions, (5)
coagulation of the cell content, and (6) inhibition of enzyme synthesis (Amensour et al. 2010; Cox
et al. 2001;Bakkali et al. 2008; Burt 2004; Di Pasqua et al. 2007; Hammer et al.2008).The mode of
myrtle extract and essential oil activity affect mainly cell wall and membrane structures. It was
reported that the permeability of bacterial cell wall and cell membrane are affected by these
extracts, leading to the release of intracellular contents outside of cell. This can be accompanied
with the disruption in the membrane function such as electron transfer, enzyme activity or nutrient
absorption (Amensour et al. 2010).Oyedemi et al. (2009) showed that essential oils
components(eugenol, _-terpineol and _-terpinene) have a bactericidal effect against the both Gram
positive and Gram negative bacteria by disrupting their membrane systems. Its important
characteristic their hydrophobicity, enables them partition the lipids of bacterial cell membrane,
disturbing the cell structure and rendering them more permeable. It has been generally reported that
the Gram negative bacteria are more resistant to the myrtle extracts and essential oils than Gram
positive (Amensour et al. 2010; Taheri et al. 2013;Kokoska et al. 2002). This resistance is likely due
to the fact that Gram negative bacteria have a wall associated with an outer complex membrane,
which slows down the passage of essential oils hydrophobic compounds (Inouye et al. 2001).
Lacking outer membrane, Gram positive bacteria are more susceptible to the myrtle extracts and
essential oils according to this model. Flavonoids are considered to possess ability to form
complexes with extracellular soluble proteins and with bacterial cell wall (Tsuchiya et al. 1996;
Cushine and Lamb 2005). Catechins, beside their versatile activities, exert antibacterial effects via
DNA gyrase inhibition. Indeed, specific binding of selected catechins was demonstrated for the N-
terminal fragment of gyrase B (Gradisaret al. 2007). Furthermore, catechins are able to restore the
susceptibility of bacterial antibiotic resistance to antibiotics such astetracycline, beta-lactams and
beta-lactamase inhibitors (Roccaroet al. 2004; Stapleton et al. 2004; Zhao et al. 2003).

5.2. Antifungal effect

The management of fungal infections possesses many problems, including a limited number of
antifungal drugs, toxicit, resistance to commonly used antifungal drugs, relapse of infections and
the high costs (Khan et al. 2003, 2010). It is therefore necessary to discover new antifungal agents
to combat the strains expressing resistance to the available antifungal drugs. One of natural products
used as therapeutic agent against fungi is myrtle, e.g. its essential oils and extracts (Table
4).Considerable myrtle extract activity was estimated by Gortziet al. (2008), while antifungal
activity of myrtle essential oil was tested against different Candida species, and minimal inhibitory
concentrations were in range 2–4 _l/ml (Cannas et al. 2013).The myrtle antifungal effect may also
be attributed to essential oil and phenolic compounds that are known to cause cell membranes
damage, causing leakage of cellular materials and ultimately the microorganism death (Cox et al.
2001). The antimicrobial, e.g. antifungal property of myrtle is suspected to be associated with their
high contents of polyphenols and oxygenated monoterpens. Modes of antifungal actions are quite
similar to those described for bacteria, concerning irreversible damage of the cell membrane and
coagulation of the cell content. Except these two, there are additional phenomena which are also
important when yeasts are considered. The first one is the establishment of a pH gradient across the
cytoplasmic membrane, and the second one is blocking of yeasts energy production which result in
disruption of the cell membrane (Djilani and Dicko 2012).

5.3. Antiviral effect

The antiviral activity of the essential oil is principally due to direct virucidal effects, by denaturing
viral structural proteins orglycoproteins (Djilani and Dicko 2012). Essential oil activity against
viruses can be dual. Proposed mechanisms suggest that essential oils eider (1) interfere with the
virus envelope by inhibiting specific processes in the viral replication cycle or (2) mask viral
components, which are necessary for adsorption or entry into host cells and cell-to-cell virus
diffusion (Saddi et al. 2007).Due to synergistic phenomena, the complex mixture of essential oils
usually shows a higher antiviral activity than individual compounds (Djilani and Dicko 2012).
Several phytochemicals have complementary and overlapping antiviral effects, which comprise
inhibition of viral nucleic acid synthesis or inhibition of other stages in viral multiplication (Jassim
and Naji 2003). The best known antiviral compound produced by many plants belonging to various
families, including M. communis L. plant, is caryophyllene (Djilani and Dicko 2012). It is
considered that further characterization of the active ingredients will reveal more useful antiviral
compounds.
5.4. Antioxidant effect

Antioxidants are compounds that react with free radicals, neutralizing them and thereby preventing
or reducing their damaging effects in the human body (Pereira et al. 2012). Lipid oxidation is also
responsible for deterioration of fats and oils resulting in change of color, flavor and nutritive value,
while oxidative stress is involved in the pathogenesis of numerous diseases (Young and
Woodside2001). In order to prevent oxidation, the addition of either synthetic or natural
antioxidants to fats, fatty foods and cosmetics is a common practice. Because of its carcinogenicity,
synthetic antioxidants used in products for human application are being restricted, considerably
increasing interest in antioxidants of natural origin (Namiki 1990; Gazzani et al. 1998; Sasaki et al.
2002; Djeridaneet al. 2006; Halliwell and Whiteman 2004).Aromatic and medicinal plants, such as
myrtle, are a source of natural antioxidants because of the activity of secondary metabolites, such as
phenylpropanoids and essential oils. These plant essential oils and extracts have been used for many
thousands ofyears in food preservation, pharmaceuticals, alternative medicineand natural therapies
(Reynolds 1996; Lis-Balchin 1997; Lis-Balchin and Deans 1997; Burt 2004). As demonstrated in
several studies, the antioxidant capacity of plant extracts is strongly related to phenolic content
(Wang et al. 1999; Wang and Stretch2001; Zheng and Wang 2003). This activity is not a property
of a single phenolic compound, but it is widely distributed among the phenolic phytochemical
constituents. Particularly anthocyans, flavonoids and phenolic acids seem to be responsible for the
antioxidant capacity (Table 2). The antioxidant activity of the phenolic compounds were attributed
to its redox properties, which allow them to act as reducing agents, hydrogen donators, sin-glet
oxygen quenchers and metal chelators (Rice-Evans et al.1995). Many in vitro studies indicate that
phenolic compounds like flavonoids and phenolic acid, can have considerable antioxidant activity
and this activity critically depends on the number and position of phenolic hydroxyls in the
aromatic ring moieties (Duthie and Crosier 2000). Generally, monophenols are less effective than
catecholic phenols, and phenolic aglicons have higher antioxidant activity than their respective
glycosides (Duthie andCrosier 2000).Several reports describe the antioxidant activities of different
extracts and compounds obtained from myrtle leaves (Chryssavgiet al. 2008; Hayder et al. 2008;
Romani et al. 2004; Rosa et al. 2008).However, leaves are not suitable for human consumption,
because of their excessive bitterness and high level of terpenic compounds. Berries on the other
hand are used to make liqueurs, jam and other food products. Ethanol extracts obtained from berries
were stud-ied according to their antioxidant properties (Alamanni and Cossu2004; Vacca et al.
2003). Tuberoso et al. (2007) found in myrtle berries very high correlation between antiradical or
antioxidant capacity and total phenol amount, but they did not investigate relative contribution of
each phenolic compound in the antioxidant capacity. On the contrary, the anthocyanine fraction
does not appear to be associated to the antioxidant capacity of myrtle berries (Angioni et al. 2011).
The use of assays that analyze these antioxidant activities evaluating the different chemical or
biological targets involved is key to elucidate mechanism involved in such activities. The
antioxidant properties and composition of theethanol extracts obtained from myrtle berries have
been studied as well (Alamanni and Cossu 2004; Montoro et al. 2006b; Vacca et al.2003) and
mainly focused on their main compounds – anthocyanins and flavonols, which are generally
considered to be responsible for antioxidant activity (Alamanni and Cossu 2004; Romani et al.
2004; Franco et al. 2002; Montoro et al., 2006a, 2006b; Tuberoso et al.2010).Various known
antioxidants like flavonoids, tannins (Romaniet al. 2004) and _-tocopherol have been isolated from
myrtle extracts. M. communis L. also exhibit the biological activities of tannins including
anticancer and antioxidant activities (Romaniet al. 2004). Nowadays, the interest in naturally
occurring antioxidants has considerably increased, because of their potential application in food,
cosmetic and pharmaceutical products, in order to replace synthetic carcinogenous, and thus
restricted antioxidants (Sasaki et al. 2002; Djeridane et al. 2006). In addition, oligomeric
proanthocyanidines, which are mainly used invascular diseases, have ability to trap lipid peroxides
and freeradicals, as well as non-competitively inhibit xanthine oxidase, which is a major generator
of free radicals (Fine 2000; Okuda2005)

5.5. Other biological effects

In addition to the biological activities of M. communis that are mentioned above, there are also
other numerous important biological activities which can be sub-classified as biochemical i.e.
pharmacological effects, including anti-inflammatory, anti-diabetic, anti-mutagenic, pro-apoptotic
activity in cancercells, cardiovascular, antiatherogenicity, activity against hepaticischemia, as well
as insecticidal, molluscicidal and protozoicidal effects. They are listed in Table 5, with indicared
references con-firming specific activity of myrtle oil, extracts or compounds. The term
pharmacological activity refers on biochemical interactions of myrtle extracts, oils or its compounds
in the bloodstream, such as interactions with different hormones and enzymes. Pharmacological
activities usually are investigated in vivo on different model organisms considering the
bioavailability of essential oils. The bioavailability represents one of the principal pharmacokinetic
and pharmacodynamics properties of drugs, and it is used to describe the fraction of unchanged
drug administered dose that reaches thesystemic circulation and can be used for a specific function
and/orstored (Djilani and Dicko 2012). It is important to notice that administered dose can be toxic
to human cells. Dell’Agli et al. (2012) demonstrated that the three different essential oils of M.
communis, Satureja thymbra and Thymus herba-barona are toxic to human cells at concentrations
of 100 _g/ml. However, this is far higher concentration than those required for the larvicidal or
mosquito-cidal effects (0.15 and 8 _g/ml, respectively). Also, extracts of M. communis L. were
found to be the most efficient against mosquito Culex pipiens with LC50 value of 16 mg/l, followed
by those of Orig-anum syriacum L., Mentha microcorphylla Koch., Pistacia lentiscus L. and
Lavandula stoechas L. (LC50 values were 36, 39, 70 and 89 mg/l,respectively) (Traboulsi et al.
2002). While extract of M. communis L. caused death of T. vaginalis at pH 4.65, but failed to do so
at pH 6.00; more efficient was Eucalyptus comaldensis extract (50 mg in 0.1 ml medium) which at
pH 5.35 caused death of T. vaginalis after 24 h (Mahdi et al. 2006). Myrtle oil exhibit antimicrobial
effects at relatively low concentrations, so its potential medical application is more possible in
comparison to essential oils obtained from some other plants (Table 4). For instance, although
peppermint oil and its components showed antimicrobial effects, its MICs values in some cases
reached concentration of 5 mg/ml (Iscan et al. 2002), being a limitation for the medicinal use of the
peppermint oil. The problem of potential toxicity also depends on the chemical composition of
myrtle extracts or essential oils used, e.g. toxic-ity of its constituting compounds. Traboulsi et al.
(2002) showed that thymol, carvacrol, (1R)-(+)-alpha-pinene and (1S)-(−)-alpha-pinene were the
most toxic (LC50 = 36–49 mg/l), while menthone, 1,8-cineole, linalool and terpineol (LC50 = 156–
194 mg/l) were less toxic. However, the fast metabolism and short half-life of these active
compounds raised the belief that there is a minimum risk of its accumulation in body tissues
(Kohlert et al. 2002). All these data are encouraging and suggest possible medical application of
extracts and essential oils of M. communis L., but yet further investigations are necessary.

6. Conclusion

Many studies have demonstrated in vitro antimicrobial and antioxidant effectiveness of M.

communis L. extracts and essential oils, which is in agreement with current trends. Myrtle seems to
be a promising plant regarding alternative antimicrobials against increasing numbers of pathogenic
microorganisms resistant to conventional antibiotic and antioxidants which should replace the
synthetic ones. However, more studies are still needed to under-stand and validate the mechanism
of action of myrtle extracts and essential oils components. Also it is important to precede work on
other extraction materials and methods because of the fact that extracting by different solvents may
indicate different compounds and consequently different extracts effects. Finally, clinical
confirmation and pharmacological standardization are required prior to their application as
antimicrobial agents and antioxidants.