Variation in antibacterial activity and chemical compositions of essential oil from different populations of myrtle

Industrial Crops and Products 61 (2014) 303–307

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Variation in antibacterial activity and chemical compositions of essential oil from different populations of myrtle Iman Bajalan a,∗ , Abdollah Ghasemi Pirbalouti b a b

Young Researchers and Elite Club, Borujerd Branch, Islamic Azad University, Borujerd, Iran Department of Medicinal Plants, Shahrekord Branch, Islamic Azad University, P.O. Box 166, Shahrekord, Iran

a r t i c l e

i n f o

Article history: Received 12 April 2014 Received in revised form 6 July 2014 Accepted 9 July 2014 Keywords: Myrtus communis L. Diversity Biological activity ␣-Pinene

a b s t r a c t Myrtle (Myrtus communis L.) belonging to the family Myrtaceae, is an evergreen shrub that grows mainly in Mediterranean climates. In Iran, the plant grows in the Zagros Mountainous Range of the country. The essential oils from the leaves and fruits of the plant are widely used to enhance the flavor of foods, and cosmetic and pharmaceutical industries. In this study, essential oil was extracted from the leaves of M. communis collected from six natural habitats in three provinces (Ilam, Lorestan, and Kermanshah), Western Iran. The hydrodistillated essential oil was analyzed by GC/MS. Results indicated that there were significant differences (p ≤ 0.05) among the various populations for the main constituents in the essential oils. The major components of the essential oils from different populations of M. communis were ␣-pinene (24.42–31.57%), limonene (trace to 23.55%), 1,8-cineole (5.92–21.21%), and linalool (8.72–11.56%). Results of the antibacterial activity in vitro indicated that the essential oils from M. communis have good inhibitory activities against bacteria, especially Staphylococcus aureus (PTCC 1112). Results obtained in this study revealed that there is a high potential of the essential oil composition variability among the populations of myrtle. The results can be used in selection programs for production of aromatic myrtle with suppressing effects on pathogens. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The development of drug resistance as well as the appearance of side effects of certain antibiotics has led to the search of new antimicrobial agents mainly among plant extracts with the goal to discover new chemical structures that overcome the above disadvantages (Lewis and Ausubel, 2006). Thus, the food industry at present uses chemical preservatives to prevent the growth of foodborne and spoiling microbes and to extend the life of foods. Mainly due to undesirable effects such as toxicity and carcinogenicity of synthetic additives, interest has considerably increased for finding naturally occurring antimicrobial compounds suitable for use in food (Feng and Zheng, 2007; Haddouchi et al., 2013). Herbs, spices, and essential oils are also well known for their various beneficial effects on human health. The use of herbs in phytotherapy is mostly due to the essential oils and their various biological activities, such as spasmolytic, carminative, hepatoprotective, antiviral, and anticarcinogenic properties (Bakkali et al., 2008; Mimica-Dukíc et al., 2010; Ghasemi Pirbalouti et al., 2014).

∗ Corresponding author. Tel.: +98 9396184417. E-mail addresses: Bajalan [email protected], [email protected] (I. Bajalan). http://dx.doi.org/10.1016/j.indcrop.2014.07.023 0926-6690/© 2014 Elsevier B.V. All rights reserved.

Myrtus communis L. (“Myrtle” in English and “Mord” in Persian), belongs to the family Myrtaceae, is an evergreen shrub that grows mainly in Mediterranean climates. (Gardeli et al., 2008; Ghasemi Pirbalouti et al., 2010). The steam or hydro-distillation are good and commonly used methods for extraction of essential oil of myrtle from the leaves, branches, fruits, and flowers (Ghasemi Pirbalouti et al., 2010). Earlier studies have been identified the chemical compositions of the essential oil from different ecotypes (Bradesi et al., 1997; Koukos et al., 2001; Flamini et al., 2004; Rahimmalek et al., 2013) that the main components of myrtle essential oils, included 1,8-cineole, ␣-pinene, limonene, and linalool. The leaves of M. communis are traditionally used as an antiseptic, disinfectant drug and hypoglycaemic agent (Elfellah et al., 1984). Different parts of the plant find various uses in the food industry, such as for flavoring meat and sauces, and in the cosmetic industry (Gardeli et al., 2008). Leaves and berries are sources of essential oil that have medicinal properties, including antimicrobial and antioxidant activities (Ghasemi Pirbalouti et al., 2010; Hayder et al., 2004; Yadegarinia et al., 2006). In Iran, myrtle grows wild in different bioclimatic zones extending from the upper semi-arid to the lower humid. Populations of M. communis grow at altitudes ranging from 900 to 1700 m, under a rainfall ranging from 400 to 600 mm/year (Ghasemi Pirbalouti, 2010).

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Table 1 Geographical properties of natural habitats of M. communis L. in Western Iran. No.

Accession name

Collection site

1 2 3 4 5 6

Ha Di MK Gh Ch Ma

Hamzeh, Lorestan Province, Iran Dinarvand, Lorestan Province, Iran Mareh khell, Kermanshah Province, Iran Ghelane Gharb, Kermanshah Province, Iran Chavar, Ilam Province, Iran Maymeh, Ilam Province, Iran

To our knowledge, no documented reports on variation of chemical composition and antibacterial activity of the essential oils from different populations of M. communis in western provinces of Iran are available. The aims of this study were (i) to determine the variation of chemical constitutes of essential oils from M. communis leaves collected from natural habitats in Western Iran, and (ii) to evaluate diversity in the antibacterial activity of the essential oils of wild populations of myrtle against two Gram-positive and two Gram-negative bacteria.

2. Materials and methods 2.1. Plant material and site description The leaves of myrtle belonging to six myrtle populations were harvested from natural habitats in the Zagros regions, western Iran in September 2013. Each sample was labeled and the location was recorded using a Global Positioning System (GPS, Vista Garmin) receiver (Table 1).

2.2. Essential oil extraction Fresh leaves were dried for 10 days at room temperature (25 ◦ C). Dried leaves were ground using food processor, and 40 g of the powdered tissue was distillated with 1 L of water for 3 h using a Clevenger-type apparatus. The collected essential oil was dried over anhydrous sodium sulphate and stored at 4 ◦ C until analyzed. The yield based on dry weight of the sample was calculated.

2.3. Gas chromatography–mass spectrometry (GC–MS) GC analysis was done on an Agilent Technologies 6890 gas chromatograph equipped with a HP-5MS 5% capillary column (30.00 m × 0.25 mm, 0.25 ␮m film thicknesses). The oven temperature was maintained at 50 ◦ C for 3 min then programmed to 290 ◦ C at 15 ◦ C/min then remind for 6 min. Helium (99.99%) was used as carrier gas at a flow rate of 1.5 ml/min, and 0.1 ␮L samples were injected manually in the split mode (50:1). Operating parameters for the EI–MS were: ionization voltage, 70 eV; interface temperature, 280 ◦ C; detector voltage, 1.66 kV; mass range, 30–450 u; scan speed, 2.86 scans/s; interval, 0.01 min (20 Hz).

2.4. Identification of components Constituents were identified by comparison of their KI (Kovats index) relative to n-alkanes (C5 –C24 ) obtained on a nonpolar HP-5 MS column by comparison of the KI, provided in the literature, by comparison of the mass spectra with those recorded by the NIST 08 (National Institute of Standards and Technology) and Willey (ChemStation Data System). Identification of oil components was accomplished based on comparison of retention times with those of authentic standards and by comparison of their mass spectral fragmentation patterns (Adams, 2007).

Latitude (UTM) 3,716,721 3,698,096 3,879,030 3,776,583 3,729,724 3678008

Longitude (UTM)

Altitude (m a.s.l)

250,198 253,851 601,413 585,867 618,499 677917

1269 761 1216 833 976 1154

2.5. Antibacterial test Isolates of bacteria strains obtained from Veterinary Medicine Faculty, Tabriz University, Iran. The antibacterial activity of the essential oils against Staphylococcus aureus (PTCC 1112), Bacillus subtilis (PTCC 1254), Klebsiella pneumonia (PTCC 1053), and Escherichia coli (PTCC 1270) were determined with the disk diffusion method (NCCLS, 2002). Briefly, bacterial suspensions were adjusted to 1.0 × 107 CFU/ml, and spread in TSA or PCA using sterile cotton swabs. Subsequently, filter paper discs (6 mm Ø; Whatman #1) were placed on the surface of Petri dishes and impregnated with 20 ␮l of essential oil. Positive controls were prepared with oxytetracycline and solphamecine, but negative control was prepared only with DMSO. After incubation at 37 ◦ C for 24 h, antibacterial activity was evaluated by measuring the radius of the inhibition zones to the nearest millimeter (Teixeira et al., 2013). Experiments were performed in triplicate at three different times. 2.6. Statistical analysis Data were analyzed with three replications using the SAS ver. 9.1 statistical software. The significance of differences among means was tested using Duncan’s multiple range test at p ≤ 0.05 level. Calculation of correlation among the compounds and antibacterial activity was performed using Minitab ver. 16. 3. Results and discussion 3.1. Chemical composition of essential oil Essential oils extracted from the populations of M. communis were analyzed by GC/MS. Generally, 27 constituents in total oil were identified from the essential oil of M. communis, which represented 90.59–96.91% of the oil (Table 2). According to the result of this study, monoterpene hydrocarbons, alcohols, and oxides were the most important of chemical groups in M. communis essential oil. The main constituents of myrtle essential oils were ␣pinene (24.42–31.57%), limonene (trace to 23.41%), 1,8-cineole (5.92–21.21%), linalool (8.72–11.56%), ␣-terpineol (7.04–8.12%), linalyl acetate (trace to 7.12%), and geranyl acetate (2.33–5.12%). Furthermore, ␣-pinene was the most abundant constituent in all samples. Limonene and 1,8-cineole were the second and third major compounds, respectively (Table 2). Similarly, the essential oils of nine populations from M. communis leaves collected from Alghero region (Northwestern Sardinia, Italy) were analyzed to evaluate variation in oil yield and the chemical composition (Mulas and Melis, 2011). They reported that the main components of the essential oils were ␣-pinene, limonene, 1,8-cineole, linalool, and geranyl-acetate. In other study by Flamini et al. (2004), the main constituents of the essential oil from myrtle collected from Italy were 1,8-cineole and ␣-pinene. In addition, Messaoud et al. (2011) reported that the main constituents of the essential oil from myrtle leaves collected from natural populations in Tunisia were ␣-pinene, camphene, and 1,8-cineole. Yadegarinia et al. (2006) reported that ␣-pinene (29.1%), limonene (21.5%),

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305

Table 2 Chemical composition of the essential oils from the leaves of M communis populations. Compounds

RIa

Ha

Di

MK

Gh

Ch

Ma

Monoterpenes hydrocarbons ␣-Thujene ␣-Pinene Camphene ␤-Pinene Myrcene ␣-Phellandrene ␦-3-Carene ␣-Terpinene Limonene (Z)-␤-Ocimene ␥-Terpinene ␣-Terpinolene

930 940 951 979 992 1006 1011 1017 1031 1036 1057 1088

0.53 26.05 0.12 0.68 tr 0.14 0.44 tr 22.90 0.14 0.50 0.36

trb 25.93 0.14 0.66 tr 0.42 0.45 tr 23.41 0.13 0.43 0.40

0.61 31.57 tr 0.81 0.29 tr 0.40 tr tr tr 0.46 0.36

0.56 28.77 0.15 0.91 0.30 0.14 0.63 0.13 tr 0.41 0.59 0.50

0.42 28.07 0.16 0.80 tr tr 0.48 tr tr 0.22 0.41 0.23

tr 24.42 0.13 0.79 0.41 tr 0.32 tr 23.55 0.11 0.49 0.38

Oxides 1,8-Cineole

1033

7.30

13.11

19.08

21.21

20.59

5.92

Alcohols Linalool Terpine-4-ol ␣-Terpineol 2,6-Octadien

1102 1173 1188 1233

11.52 0.69 8.12 7.44

10.10 0.81 7.04 1.10

11.56 0.52 7.16 tr

11.26 0.74 7.64 0.32

8.72 0.50 7.41 0.42

11.25 0.29 7.54 tr

Esters Linalyl acetate (Z)- Pinocarveol Myrtenyl acetate ␣-Terpinyl acetate Neryl acetate Geranyl acetate

1252 1304 1330 1343 1368 1379

tr tr 0.11 tr 0.28 2.51

tr 0.12 tr tr 0.27 2.37

7.23 tr 0.23 3.65 tr 2.33

6.17 0.23 tr 4.41 0.23 4.88

6.00 0.14 tr 4.95 0.31 4.60

7.57 tr tr 4.43 0.45 5.12

Phenylpropene Methyl eugenol

1399

2.97

3.23

tr

1.48

1.95

0.12

Sesquiterpene hydrocarbons ␤-Caryophyllene ␣-Humulene

1412 1446

1.70 0.97

1.35 0.92

1.88 1.73

1.50 1.30

1.36 1.11

1.22 1.02

1574

0.92 51.86 7.30 27.77 2.90 2.97 2.67 0.92

1.32 51.97 13.11 19.05 2.76 3.23 2.27 1.32

1.32 34.50 19.08 19.24 13.44 0.00 3.61 1.32

1.57 33.09 21.21 19.96 15.92 1.48 2.80 1.57

1.74 30.79 20.59 17.05 16.00 1.95 2.47 1.74

1.38 50.60 5.92 19.08 17.57 0.12 2.24 1.38

96.39

93.71

91.19

96.03

90.59

96.91

Oxygenated sesquiterpenes Caryophyllene oxide Total monoterpene hydrocarbons Total oxides Total alcohols Total esters Total phenylpropene Total sesquiterpene hydrocarbons Total oxygenated sesquiterpenes Total (%)

% GC peak, the percentage composition was computed from the GC peak areas. a Retention indices (RI) relative to C5-C24 n-alkanes on apolar HP-5 MS column. b tr: <0.1.

1,8-cineole (17.9%), and linalool (10.4%) were the major compositions of the essential oil extracted from an ecotype of myrtle in Iran. In a study, Rasooli et al. (2002) reported that the main constituents of the essential oil from myrtle leaves cultivated at botanical garden (Tehran) Iran were ␣-pinene (29.4%), limonene (21.2%), 1,8-cineole (18%), linalool (10.6%), linalyl acetate (4.6%), and ␣-terpineole (3.1%). In addition, another investigation by Mahboubi and Ghazian Bidgoli (2010) the identified major components in essential oil of myrtle cultivated at Kashan, Central Iran were 1,8-cineole (36.1%), ␣-pinene (22.5%), linalool (8.4%), bornyl acetate (5.2%), ␣-terpineol (4.4%), linalyl acetate (4.2%), and limonene (3.8%). Ghasemi Pirbalouti et al. (2014) reported high levels of oxygenated monoterpenes (24.7–66.9%), including 1,8-cineole, linalool, and ␣-terpineol, followed by hydrocarbon monoterpenes (22.3–58.5%), including ␣-pinene and limonene in the essential oil from various populations collected from southwestern Iran (Khuzestan and Lorestan provinces). Rahimmalek et al. (2013) identified diversity among the main chemical constituents in the essential oils from 21 wild M. communis populations collected from different geographical regions in southwestern of Iran. They have introduced two chemotypes

for Iranian myrtle populations, including ␣-pinene/1,8-cineole, and limonene/␣-pinene.

3.2. Antibacterial activity The antibacterial activity of the essential oils against four bacteria was evaluated using the disk diffusion method (Fig. 1). The results indicated that the essential oils of the investigated populations had a significant difference (p ≤ 0.05) for antibacterial activity against the tested bacteria. The highest antibacterial activity was observed against S. aureus with the strongest inhibition zones (32.66 mm) for the essential oil from the Ch population, while the essential oil from the Di population exhibited weak antibacterial activity against S. aureus (15.33 mm) (Fig. 1). These results were in agreement with results of a study by Yadegarinia et al. (2006). In addition, Alem et al. (2008) reported that the myrtle extract had the antibacterial effect against S. aureus (MIC = 0.5 mg/ml). Our results revealed that S. aureus (Gram-positive) was more sensitive to the essential oils of myrtle than E. coli (Gram-negative). Results of other studies (Bouzouita et al., 2003; Gündüz et al., 2009; Ghasemi

32.6a

25.3c 24.6cd 21.6e 24.6cd

23.3d

E. coli K. pneumoniae B. subtilis

6n 6n 6n 6n

6n 6n

8.6jklm 10.3hi 6n

6n 8.3lm 8.6jklm

8.6lkm 10.3hi 6n

10hij 10.8h 6n

10

15.3f 9ijkl 9.6hijk 12.3g

20 15

23.6d

30b

S. aureus

25

7.3nm 8lm 10.6h

Inhabition zone (mm)

30

30b

35

29.6b

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28.6b

306

5 0

Essential oils of myrtle populations and controls Fig. 1. Antibacterial activity of the essential oils from M. communis leaves collected from different natural habitats and control groups (positive and negative). Means with different letter in a row are statistically significant at 5% level probability. Abbreviations of populations: Ha: Hamzeh; Di: Dinarvand, Mk: Mareh khell, Gh: Ghelane Gharb, Ch: Chavar, Ma: Maymeh.

Table 3 Simple correlation between the main components of the essential oil from different populations of myrtle leaves. No.

Components

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8

␣-Pinene Limonene 1,8-Cineole Linalool ␣-Terpineol Linalyl acetate ␣-Terpinyl acetate Geranyl acetate

1.000 −0.862* 0.805* 0.080 −0.318 0.404 0.349 −0.238

1.000 −0.928** 0.213 0.221 −0.602 −0.676 −0.235

1.000 −0.372 −0.408 0.377 0.487 0.116

1.000 0.358 0.051 −0.184 −0.220

1.000 −0.210 −0.143 0.182

1.000 0.951** 0.636

1.000 0.776*

1.000

* **

Significant at 5% probability level. Significant at 1% probability level.

Pirbalouti et al., 2014) indicate antibacterial effects of the essential oil from myrtle against some bacteria. In general, the essential oil of various populations of myrtle had higher antibacterial activity against the tested bacteria. This property could be resulted from the relatively high amount of monoterpenes (␣-pinene and 1,8-cineole) in the essential oils of various populations. Major or trace compounds in the oil might give rise to the antimicrobial activity exhibited. Possible synergistic and antagonistic effect of compounds in the oil should also need to be taken into consideration (Lopes-Lutz et al., 2008). Different components of essential oils can interact to either reduce or increase antimicrobial efficacy (Delaquis et al., 2012). The interaction between essential oil compounds can produce four possible types of effects: indifferent, additive, antagonistic, or synergistic effects (Pei et al., 2009; Delgado et al., 2004). It has been reported that essential oils containing aldehydes or phenols, such as cinnamaldehyde, citral, eugenol, carvacrol or thymol as major components showed the highest antibacterial activity, followed by essential oils containing terpene alcohols (Sachetti et al., 2005; Bassolé and Juliani, 2012). 3.3. Correlation between the main compounds Results of correlation analysis among the major components of M. communis essential oils indicted that the highest correlation coefficients obtained from the correlation between linalyl acetate and ␣-terpinyl acetate (0.951), limonene and 1,8-cineole (−0.928), ␣-pinene and limonene (−0.862), ␣-pinene and 1,8-cineole (0.805), and ␣-terpinyl acetate and geranyl acetate (0.776) (Table 3). Probably in positive correlations, these compounds are double-bond

isomers or are both produced from a pathway biosynthesis (Ghasemi Pirbalouti, 2010). Similarly, results of correlation analysis of essential oil of myrtle by Rahimmalek et al. (2013) showed that correlations between ␣-pinene and 1,8-cineole (+0.90), 1,8-cineole and limonene (−0.90), limonene and ␣-pinene (−0.87), linalool and linalyl acetate (0.58), and ␣-terpineol and limonene (0.50). 4. Conclusions The main constituents of the essential oil from the myrtle leaves were ␣-pinene, limonene, 1,8-cineole, linalool, ␣-terpineol, linalyl acetate, and geranyl acetate. Percentage of ␣-pinene, as a main constituent in the essential oil from myrtle ranged from 24.42% in the Ma population to 31.57% in the MK population. The highest positive correlation was between linalyl acetate and ␣-terpinyl acetate (+0.951) and ␣-pinene and 1,8-cineole (+0.805), while the highest negative correlation was between 1,8-cineole and limonene (−0.928). In addition, the essential oil of M. communis is effective for inhibition or control of bacteria strains, especially S. aureus. The essential oil from the studied populations of M. communis could be used as a natural antibacterial agent, especially against S. aureus. The present study indicates that essential oil components of wild populations of myrtle vary with environmental conditions, and geographic origin. Essential oils of myrtle leaves were characterized by high levels of oxygenated monoterpenes and hydrocarbon monoterpenes. Monoterpenes were the main constituents of the essential oil of the leaves of the collected myrtle. In final, diversity in essential oil composition and antibacterial activity of myrtle can result from genetic diversity and differences in environmental conditions and their interactions.

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