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Int. J. Mol. Sci. 201213, 16580-16591; doi:10.3390/ijms131216580 

 

International Journal of 



Molecular Sciences

 

ISSN 1422-0067 

www.mdpi.com/journal/ijms 



Article 

Chemical Composition and Biological Activities of the Essential 

Oils from Three Melaleuca Species Grown in Tunisia 

Ismail Amri 

1

, Emilia Mancini 

2

, Laura De Martino 

2

, Aurelio Marandino 

2

, Hamrouni Lamia 

1



Hanana Mohsen 

3

, Jamoussi Bassem 

4

, Mariarosa Scognamiglio 

5

, Ernesto Reverchon 

5

 and 

Vincenzo De Feo 

2,

Laboratory for Forest Ecology, National Institute for Research in Rural Engineering,  



Water and Forests, BP 10, 2080 Ariana, Tunisie; E-Mails: amri_amri@live.fr (I.A.);  

hamrounilam@yahoo.fr (H.L.) 

2

  Department of Pharmaceutical and Biomedical Sciences, University of Salerno,  



Via Ponte Don Melillo, 84084 Fisciano (Salerno), Italy; E-Mails: emancini@unisa.it (E.M.);  

ldemartino@unisa.it (L.M.); aureliomarandino@libero.it (A.M.)  

3

  Plant Molecular Physiology Laboratory, Center of Biotechnology of Borj-Cèdria, BP 901,  



2050 Hammam-Lif, Tunisie; E-Mail: punto80@yahoo.com  

Chemistry Laboratory, Higher Institute of Education and Continuous Training,  



43 Rue de la Liberté, 2019 Le Bardo, Tunisie; E-Mail: jamoussi_bassem@yahoo.fr 

5

  Department of Industrial Engineering, University of Salerno, via Ponte Don Melillo,  



84084 Fisciano (Salerno), Italy; E-Mails: mrscogna@unisa.it (M.S.); ereverchon@unisa.it (E.R.) 

*  Author to whom correspondence should be addressed; E-Mail: defeo@unisa.it;  

Tel.: +39-089-969-751; Fax: +39-089-969-602. 



Received: 25 September 2012; in revised form: 25 October 2012 / Accepted: 29 November 2012 /  

Published: 5 December 2012 

 

Abstract: The chemical composition of the essential oils of Melaleuca armillaris Sm., 



Melaleuca styphelioides Sm. and Melaleuca acuminata F. Muell., collected in Tunisia, was 

studied by means of GC and GC-MS analysis. In all, 46 compounds were identified, 38 for 



M. armillaris, 20 for M. acuminata and eight for M.  styphelioides, respectively. The 

presence of a sesquiterpenic fraction (52.2%) characterized the oil from M. armillaris;  



M. sthypheliodes oil was rich in methyl eugenol, a phenolic compound (91.1%), while  

M. acuminata oil  is mainly constituted by oxygenated monoterpenoids (95.6%). The 

essential oils were evaluated for their in vitro potentially phytotoxic activity against 

germination and initial radicle growth of Raphanus sativus L.,  Lepidium  sativum  L., 

Sinapis arvensis L., Triticum durum L. and Phalaris canariensis L. seeds. The radicle 

OPEN ACCESS


Int. J. Mol. Sci. 201213 16581 

 

 

elongation of five seeds was inhibited at the highest doses tested, while germination of all 



seeds was not affected. Moreover, the essential oils showed low antimicrobial activity 

against eight selected microorganisms.  



Keywords:  Melaleuca acuminata;  Melaleuca armillaris;  Melaleuca styphelioides

phytotoxic activity; antimicrobial activity 

 

1. Introduction 

The genus Melaleuca  (family Myrtaceae, subfamily Leptospermoideae) consists of about  

230 species rich in volatile oils. Several studies demonstrated the efficacy of some Melaleuca essential 

oils against different types of bacteria and fungi [1–4], as in the case of Melaleuca alternifolia essential 

oil against methicillin-resistant Staphylococcus aureus (MRSA), Legionella pneumophila,  and 

Staphylococcus aureus [5].  

Essential oils are becoming more popular, because many synthetic drugs are connected with 

unpleasant side effects, such as nephrotoxicity or ototoxicity. Volatile oils also represent an interesting 

alternative due to an emerging resistance of microorganisms against synthetic agents. Essential oils can 

exert not only bacteriostatic and bactericidal effects, but also demonstrate activity against fungi and 

yeasts [5]. 

Moreover, in the past, it seemed that certain Melaleuca species may have allelopathic properties, 

resulting in an inhibition of other species in the same ecosystem [6]. The bare ground in Melaleuca 

forests was reported as an example of allelopathy in this genus [7]. 

Melaleuca armillaris Sm. is one of the most widely cultivated melaleucas. It is commonly known as 

Bracelet Honey Myrtle and grows into a large spreading shrub or small tree. Literature reports about 



M. armillaris remain scarce. GC/MS investigations of its  essential oil  revealed the presence of  

1,8-cineole as the main component [8–12]. Only little information could be found in the literature 

about the composition of Melaleuca styphelioides Smoils. Farag and coworkers [12] reported that the 

essential oil of this species contained mainly caryophyllene oxide (43.8%) and (−)-spathulenol (9.7%). 

Previously, the same authors [13] reported that the essential oil of M. styphelioides contained mainly 

caryophyllene (49.9%) and methyl eugenol (26.6%). Only a few reports are available about  



Melaleuca acuminata F. Muell. [14,15].  

In continuation of our studies on the possible phytotoxic and antimicrobial activity of essential oils 

from plants collected in the Mediterranean area [16,17], we studied the chemical composition of the 

essential oils from Melaleuca armillaris, M. acuminata and M. styphelioides and their possible in vitro 

effects against germination and initial radicle elongation of Raphanus sativus L. (radish),  

Lepidium sativum L. (garden cress), Sinapis arvensis L. (wild mustard), Triticum durum L. (wheat)  

and Phalaris canariensis L. (canary grass) and the antimicrobial activity against eight 

 

selected microorganism.  



Int. J. Mol. Sci. 201213 16582 

 

 

2. Results and Discussion 



2.1. Chemical Composition of the Essential Oils  

Hydrodistillation yielded 0.65%, 0.53% and 0.35% of essential oil (on a dry mass basis) for  



M. armillaris, M. acuminata and  M. stypheliodes,  respectively. Table 1 shows the chemical 

composition of the three Melaleuca  oils; compounds are listed according to their elution order on a 

HP-5MS column. In all, 46 compounds were identified, 38 for M. armillaris, accounting for 99.3% of 

the total oil, 20 for M. acuminata, accounting for 99.7% of the total oil, and eight for M. styphelioides 

(92.4%), respectively.  

Table 1. Percent composition of Melaleuca armillaris,  Melaleuca acuminata and 

Melaleuca styphelioides essential oils. 

Compound RI 

a

 RI 

b

 

Melaleuca 

armillaris 

Melaleuca 

acuminata 

Melaleuca 

styphelioides 

Identification 

c

 

α-Pinene 935 

1032 





0.1 

1,2,3 


Myrcene 989 

1162 




1,2,3 

p-Cymene 1017 

1269 


0.1 



1,2,3 

1,8-Cineole 1022 

1213 

3.6 


11.7 

1,2,3 



Perillene 1104 

1429 


0.1 


1,2 


α-Campholenal 1117 

1497 


0.7 -  1,2 

Nopinone 1124 

1597 


0.4 

3.0 


1,2 


trans-Pinocarveol 1127 

1664 


6.9 25.1  - 

1,2 


Camphor 1131 

1532 


1.2 


1,2,3 


1-Terpineol 1133 

 



0.5 

1,2 



trans-Sabinol 1136 

1720 


0.6 

1.6 


- 1,2 

trans-Verbenol 1139 

1683 


0.4 

1.1 -  1,2 

Eucarvone 1150 

1932 


1.4 

6.4 


1,2 


Isoborneol 1155 

1633 


0.5 

0.3 


1,2 


Viridene 1160 

 

1.3 



2.4 

1,2 



Terpinen-4-ol 1168 

1611 


0.4 

- - 1,2 


m-Cymen-8-ol 1179 

 

0.2 



- -  1,2 

p-Cymen-8-ol 1180 

1856 


0.1 

- -  1,2 

α-Terpineol 1182 

1706 


7.7 



1,2,3 

Dihydrocarveol 1183 

1755 

9.0 


23.6 -  1,2 

Myrtenol 1187 

1804 

6.4 


12.3 

1,2 



Myrtenal 1191 

1648 


0.8 



1,2 

cis-Dihydrocarvone 

1194 1620 

0.5 





1,2 

Verbenone 1196 

1723 

3.7 


7.8 

1,2 



p-Cymen-9-ol 1200 

 

0.1 



0.2 

-  1,2 


9-Decen-1-ol 1260 

 

0.6 



- 1,2 


Citronellyl acetate 

1353 


1662 

0.5 


1,2 



cis-Piperitone oxide 

1360 


1733 

0.8 


1,2 



Methyl eugenol 

1401 


2016 



91.1 

1,2 


4,8-β-epoxy-Caryophyllane 

1424  


0.4 



1,2 

cis-α-Ambrinol 1435 

 

0.6 



-  -  1,2 

Int. J. Mol. Sci. 201213 16583 

 

 

Table 1. Cont. 



Compound RI 

a

 RI 

b

 

Melaleuca 

armillaris 

Melaleuca 

acuminata 

Melaleuca 

styphelioides 

Identification 

c

 

allo-Aromadendrene 

1446 1661 



0.2 



1,2 

Aromadendrene 1453 

1628 

4.1 -  -  1,2 



γ-Himachalene 1483 

 

1.5 



- -  1,2 

10-Undecen-1-ol, acetate 

1495 

 

0.6 



1,2 



cis-Calamenene 1511 

 

19.0 



-  -  1,2 

Germacrene B 

1539 

1854 


1.8 

0.6 


1,2 


Spathulenol 1562 

2150 


4.4 

0.3 


0.4 

1,2 


Caryophyllene oxide 

1565 


2008 

0.8 


0.6 

0.4 


1,2,3 

Globulol 1568 

2098 

1.2 


1,2 



Guaiol 1575 

2108 


1.3 



1,2 

β-Atlantol 1586 

 

0.5 


0.1 


1,2 

1-epi-Cubenol 1591 

2088 

0.5 


0.2 

-  1,2 


Torreyol 1615 

 

15.1 



1,2 



γ-Eudesmol 1623 

2173 


0.7 



1,2 

Methyl jasmonate 

1628 

 

0.6 



1,2 



α-Eudesmol 1633 

2250 


0.3 



1,2 

Total compounds  

 

 

99.3 



99.7 

92.4 

 

Monoterpene hydrocarbons 



 

 

- - 0.2 

 

Oxygenated Monoterpenes  



 

 

44.6 95.6 



 

Sesquiterpene hydrocarbons 



 

 

26.4 0.6  0.2 

 

Oxygenated Sesquiterpenes 



 

 

25.2 1.1  0.9 

 

Phenolic compound 



 

 

- - 91.1   

Carbonylic compounds 

 

 



1.2 -  - 

 

Non terpenes 



 

 

1.3 2.4  - 

 

Others  


 

0.6 -  - 

 

a



 Kovats retention index on HP-5 MS column; 

b

 Kovats retention index on HP Innowax; 



c

 1 = Kovats 

retention index, 2 = mass spectrum, 3 = co-injection with authentic compound; t = trace, less than 0.05%.  

In the oil from M. armillaris, the oxygenated monoterpenoids amounted to 44.6%; on the other 

hand, the total sesquiterpenic fraction amounted to 51.6% of the total oil. The main compounds are the 

sesquiterpene cis-calamenene  (19.0%) and the oxygenated sesquiterpene torreyol (15.1%). Other 

compounds, in a lesser amount, are dihydrocarveol (9.0%) and α-terpineol (7.7%), both oxygenated 

monoterpenoids. In the literature, Chabir and coworkers [8,9] reported 1,8-cineole (85.8%) as the most 

abundant compound in M. armillaris. The major compound in the essential oil of M. styphelioides was 

methyl eugenol (91.1%), a phenolic compound. In the literature, Farag and coworkers [12] reported 

that the essential oil of this species contained mainly 

caryophyllene oxide (43.8%), followed by  

(−)-spathulenol (9.7%). In another paper [13], the same authors reported that the essential oil of this 

species contained mainly caryophyllene (50.0%) and methyl eugenol (26.6%). 

In the oil from M. acuminata, the oxygenated monoterpenoids amounted to 95.6%, with a total 

sesquiterpenes amount of 1.7% (0.6% sesquiterpene hydrocarbons and 1.1% of oxygenated 

sesquiterpenes) of the total oil. trans-Pinocarveol (25.1%), dihydrocarveol (23.6%), myrtenol (12.3%) 

and 1,8-cineole (11.7%) were the most abundant among the oxygenated monoterpenes.  



Int. J. Mol. Sci. 201213 16584 

 

 

In the literature, only two references reported the chemical composition of the essential oil of  



M. acuminata: Smith and coworkers [14,15] reported that cineole was the main component of the 

essential oil of M. acuminata.  



2.2. Phytotoxic Activity 

The three essential oils were evaluated for their phytotoxic activity against germination and radicle 

elongation (Table 2) of radishes and garden cress, two species frequently utilized in biological assays, 

and of wild mustard, wheat and canary grass, three weed species.  



Table 2. Phytotoxic activity of the essential oils of Melaleuca armillaris,  

Melaleuca styphelioides and Melaleuca acuminata against germination and radicle 

elongation of Sinapis arvensis,  Triticum  durum,  Phalaris  canariensis,  Raphanus  sativus 

and Lepidium sativum, 120 h after sowing. Data are expressed in centimeter. 

Sinapis arvensis Germinated seeds ± SD (cm) 

Sinapis arvensis Radicle elongation ± SD (cm) 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Control 


8.8 ± 0.8 

8.8 ± 0.8 

8.8 ± 0.8 

Control 


1.4 ± 0.7 

1.4 ± 0.7 

1.4 ± 0.7 

0.062 μg/mL 

9.2 ± 0.6 

9.2 ± 0.6 

9.8 ± 0.6 

0.062 μg/mL 

1.2 ± 0.7 

1.3 ± 0.6 

1.9 ± 0.8 ** 

0.125 μg/mL 

8.3 ± 1.4 

9.9 ± 0.6 

9.4 ± 0.6 

0.125 μg/mL 

1.3 ± 0.5 

1.4 ± 0.6 

1.5 ± 0.9 

0.25 μg/mL 

8.5 ± 1.6 

9.2 ± 0.6 

9.8 ± 0.6 

0.25 μg/mL 

1.0 ± 0.4 

1.1 ± 0.5 

1.4 ± 0.8 

0.625 μg/mL 

7.9 ± 1.4 

9.9 ± 0.6 

10.0 ± 0.0 *  0.625 μg/mL 

2.3 ± 1.3 * 

1.0 ± 0.6 

1.1 ± 0.4 

1.25 μg/mL 

9.2 ± 0.6 

9.9 ± 0.6 

9.8 ± 0.6 

1.25 μg/mL 

1.7 ± 1.0 

1.3 ± 0.9 

1.0 ± 0.6 ** 

2.5 μg/mL 

8.8 ± 1.2 

9.5 ± 1.1 

9.8 ± 0.6 

2.5 μg/mL 

1.0 ± 0.4 

1.2 ± 0.7 

0.9 ± 0.6 ** 



Triticum durum Germinated seeds ± SD (cm) 

Triticum durum Radicle elongation ± SD (cm) 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca  

acuminata 

Control 


8.3 ± 0.9 

8.3 ± 0.9 

8.3 ± 0.9 

Control 


4.4 ± 2.3 

4.4 ± 2.3 

4.4 ± 2.3 

0.062 μg/mL 

8.7 ± 1.2 

9.1 ± 1.3 

8.0 ± 1.4 

0.062 μg/mL 

4.7 ± 2.3 

4.7 ± 1.9 

4.0 ± 1.7 

0.125 μg/mL 

8.7 ± 0.6 

7.4 ± 3.4 

8.6 ± 1.1 

0.125 μg/mL 

4.5 ± 2.2 

4.8 ± 2.2 

4.7 ± 1.9 

0.25 μg/mL 

7.3 ± 1.5 

8.0 ± 1.3 

7.7 ± 1.1 

0.25 μg/mL 

5.7 ± 1.6 * 

5.3 ± 1.7 

4.4 ± 2.6 

0.625 μg/mL 

8.7 ± 0.6 

8.0 ± 2.3 

8.0 ± 1.1 

0.625 μg/mL 

3.2 ± 1.9 * 

5.1 ± 1.8 

4.2 ± 2.0 

1.25 μg/mL 

6.7 ± 3.5 

9.9 ± 1.1 

7.7 ± 0.6 

1.25 μg/mL 

3.9 ± 1.5 

4.9 ± 1.8 

2.8 ± 1.7 ** 

2.5 μg/mL 

8.7 ± 1.5 

9.6 ± 1.7 

8.6 ± 1.1 

2.5 μg/mL 

3.8 ± 1.7 

4.0 ± 1.8 

3.4 ± 2.0 

Phalaris canariensis Germinated seeds ± SD (cm) 

Phalaris canariensis Radicle elongation ± SD (cm) 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Doses 

Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Control 


8.8 ± 0.9 

8.8 ± 0.9 

8.8 ± 0.9 

Control 


3.1 ± 1.2 

3.1 ± 1.2 

3.1 ± 1.2 

0.062 μg/mL 

8.8 ± 2.2 

9.0 ± 0.6 

8.7 ± 1.0 

0.062 μg/mL 

2.8 ± 1.1 

3.2 ± .3 

2.8 ± 0.7 

0.125 μg/mL 

9.2 ± 1.6 

9.7 ± 0.0 

9.7 ± 0.0 

0.125 μg/mL 

3.0 ± 1.4 

3.3 ± 1.1 

3.4 ± 1.2 

0.25 μg/mL 

9.4 ± 0.6 * 

8.1 ± 1.1 

8.7 ± 1.0 

0.25 μg/mL 

2.4 ± 1.2 

3.7 ± 1.3 

3.2 ± 1.5 

0.625 μg/mL 

9.2 ± 0.6 

8.7 ± 1.0 

9.4 ± 0.6 

0.625 μg/mL 

2.6 ± 1.2 

3.9 ± 1.0 

2.5 ± 0.9 * 

1.25 μg/mL  10.6 ± 0.0 ** 

8.7 ± 1.7 

8.4 ± 1.4 

1.25 μg/mL 

2.6 ± 0.8 

3.2 ± 1.3 

1.6 ± 1.4 *** 

2.5 μg/mL 

9.2 ± 0.6 

8.4 ± 0.6 

8.1 ± 1.5 

2.5 μg/mL 

3.5 ± 1.6 

2.8 ± 1.1 

2.7 ± 0.7 



Int. J. Mol. Sci. 201213 16585 

 

 

Table 2. Cont. 



Raphanus sativus Germinated seeds ± SD (cm) 

Raphanus sativus Radicle elongation ± SD (cm) 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Doses 

Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Control 


9.3 ± 0.7 

9.3 ± 0.7 

9.3 ± 0.7 

Control 


6.1 ± 2.8 

6.1 ± 2.8 

6.1 ± 2.8 

0.062 μg/mL 

10.0 ± 0.0 

9.6 ± 0.0 

9.0 ± 0.6 

0.062 μg/mL 

6.7 ± 3.3 

6.0 ± 2.5 

4.8 ± 2.4 

0.125 μg/mL 

10.0 ± 0.0 

8.9 ± 0.6 

10.1 ± 0.6  0.125 μg/mL 

6.0 ± 3.4 

4.0 ± 2.4 ** 

5.0 ± 2.2 

0.25 μg/mL 

10.0 ± 0.0 

9.9 ± 0.6 

9.4 ± 0.0 

0.25 μg/mL 

6.4 ± 3.2 

5.3 ± 2.4 

4.5 ± 2.3 

0.625 μg/mL 

9.7 ± 0.6 

9.6 ± 0.0 

9.7 ± 1.1 

0.625 μg/mL 

7.1 ± 2.9 

5.1 ± 2.9 

4.4 ± 2.4 

1.25 μg/mL 

9.7 ± 0.6 

8.9 ± 0.6 

8.6 ± 1.6 

1.25 μg/mL 

6.1 ± 2.9 

4.0 ± 2.1 ** 

3.5 ± 2.0 *** 

2.5 μg/mL 

9.7 ± 0.6 

9.3 ± 0.6 

9.7 ± 1.6 

2.5 μg/mL 

6.3 ± 2.9 

4.8 ± 2.6 

4.2 ± 2.4 * 



Lepidum sativum Germinated seeds ± SD (cm) 

Lepidum sativum Radicle elongation ± SD (cm) 

Doses 


Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Doses 

Melaleuca 

armillaris 

Melaleuca 

styphelioides 

Melaleuca 

acuminata 

Control 


8.2 ± 0.5 

8.2 ± 0.5 

8.2 ± 0.5 

Control 


2.8 ± 1.8 

2.8 ± 1.8 

2.8 ± 1.8 

0.062 μg/mL 

8.2 ± 0.0 

8.2 ± 0.6 

9.2 ± 1.0 

0.062 μg/mL 

2.7 ± 1.5 

1.5 ± 1.1 *** 

2.7 ± 2.1 

0.125 μg/mL 

8.9 ± 1.5 

8.7 ± 0.6 

8.2 ± 1.0 

0.125 μg/mL 

2.5 ± 1.2 

1.61.1 *** 

2.7 ± 1.5 

0.25 μg/mL 

9.5 ± 0.6 

8.5 ± 1.6 

9.2 ± 1.0 

0.25 μg/mL 

2.3 ± 2.2 

1.8 ± 1.4 *** 

2.7 ± 2.1 

0.625 μg/mL 

8.5 ± 0.6 

8.5 ± 0.9 

9.2 ± 1.0 

0.625 μg/mL 

2.8 ± 1.4 

1.1 ± 1.0 *** 

2.7 ± 2.1 

1.25 μg/mL 

9.2 ± 1.0 

7.5 ± 0.9 

8.9 ± 1.5 

1.25 μg/mL 

1.9 ± 1.4 

1.8 ± 1.1 ** 

2.2 ± 1.8 

2.5 μg/mL 

7.2 ± 1.0 

8.7 ± 0.6 

7.9 ± 1.5 

2.5 μg/mL 

2.9 ± 1.4 

1.9 ± 1.0 ** 

2.6 ± 2.4 

*  p < 0.05; ** p < 0.01; *** p  < 0.001 vs. control. Results are the mean ± standard deviation (SD) of  

three experiments. 

The oils seem to be ineffective against germination, but they affected the radicle elongation of the 

five tested seeds. The essential oil of M. styphelioides, at all doses tested, significantly inhibited the 

radicle elongation of garden cress. The radicle elongation of wild mustard and radish were inhibited  

by  M. acuminata oil at the highest doses (2.5 μg/mL, 1.25 μg/mL) used. At doses of 1.25 and  

0.625 μg/mL, the essential oil of M. acuminata significantly inhibited the radicle elongation of canary 

grass (Table 2). The difference in biological activity of the oils could be attributed to their different  

chemical composition. 

On the other hand, the oil of M. acuminata was rich in oxygenated monoterpenoids,  

trans-pinocarveol, dihydrocarveol, myrtenol and 1,8-cineole. trans-Pinocarveol was reported as one of 

the main components of the strong phytotoxic oil from a Cistus ladanifer L. population [18]. 

Yatagai and coworkers [19] reported that the leaf oil of Melelauca bracteata F. Muell. had the 

strongest germination and growth-inhibition activity against radish seeds. 

The roots were probably more sensitive than shoots to the phytotoxic activity of the oil; the process 

of germination was active while the oil probably affected the elongation process. Such activity of the 

essential oils could help to explain the ecological role of the genus Melaleuca in the Mediterranean area.  

2.3. Antimicrobial Activity 

The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration 

(MBC) values of the essential oils against eight selected microorganisms are reported in Table 3.  


Int. J. Mol. Sci. 201213 16586 

 

 

Table 3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal 

Concentration (MBC) Values (μg/mL) of essential oils from the three Melaleuca Species 

and MIC of the reference antibiotic, chloramphenicol. Results are the mean of  

three experiments. 

Bacterial strain 

Melaleuca 

styphelioides 

Melaleuca 

armillaris 

Melaleuca 

acuminata 

Chloramphenicol 

MIC 

a

 MBC 

b

 MIC  MBC  MIC  MBC 

 

Bacillus subtilis 

ATCC 6633 

100 >100 50 100 50  n.a. 

12.5 


Staphylococcus aureus 

ATCC 25923 

100 >100 100 >100 50  100 

25 


Staphylococcus 

epidermidis 

ATCC 12228 

50 100 50 n.a. 

12.5 


n.a. 

3.12 


Streptococcus faecalis 

ATCC 29212 

>100 n.a. >100 n.a. 100 100 

25 


Escherichia coli 

ATCC 25922 

100 >100 50 n.a. 25  50 

12.5 


Klebsiella pneumoniae 

ATCC 10031 

>100 n.a. 100 >100 100 >100 

50 


Proteus vulgaris 

ATCC 13315 

>100 n.a.  50 100 50  100 

25 


Pseudomonas aeuriginosa 

ATCC 27853 

>100  n.a.  >100 n.a. >100  n.a. 

100 


a

 MIC, Minimal Inhibitory Concentration (μg/mL); 

b

 MBC, Minimal Bactericidal Concentration (μg/mL); 



n.a., not active. 

The essential oils showed inhibitory activity against the gram-positive pathogens, among which  

is  S. epidermidis. Among gram-negative bacteria, E. coli was affected by the oil of 

 

M. armillaris and M. acuminata. The essential oil of M. acuminata was more active than other oils and 

presumably this activity is related to the high amounts of oxygenated monoterpenoids. 

Farag and coworkers [12,13] reported the effect of the essential oil from the leaves of M. armillaris



M. ericifolia (Smith), M. leucadendron (Linn.) and M. styphelioides against the growth of some 

microorganisms. The results demonstrated that the degree of the microbial inhibition is largely 

dependent on the species. 

M. ericifolia exhibited the highest inhibitory effects against Bacillus subtilis 

and Aspergillus niger.

 The antimicrobial properties of Melaleuca essential oil have been reported in 

several studies [20] Terpinen-4-ol is considered to be the principal active component of  



Melaleuca alternifolia Cheel (tea tree) oil [21–23]. Terpinen-4-ol could constitute an interesting 

alternative in the therapy of MRSA infections of the skin [5]. In the literature, it was reported that 

some members of the Myrtaceae family (Eucalyptus globulus and Melaleuca alternifolia) whose 

essential oil consisted mainly of monocyclic and bicyclic monoterpenes (e.g., 1,8-cineole and terpinen-4-ol) 

effectively inhibited the growth of drug-resistant bacterium strains [5].  


Int. J. Mol. Sci. 201213 16587 

 

 

3. Experimental Section  



3.1. Plant Material 

Leaves of Melaleuca armillarisM. acuminata and M. styphelioides Sm. were collected from the 

Botanical Garden of the National Institute of Researches on Rural Engineering, Water and Forests 

(Ariana, Tunisia) in April 2011. Five samples were collected from more than five different trees, 

mixed for homogenization and used in three replicates for essential oil extractions. Specimens were 

identified by Dr. H. Lamia, and voucher specimens were deposited at the herbarium of the Laboratory 

of Forestry Ecology at the National Institute of Research on Rural Engineering, Water and Forest (Tunisia).  

3.2. Isolation of the Volatile Oils 

One hundred grams of dried leaves of each Melaleuca species were ground in a Waring blender and 

then, subjected to hydrodistillation for 3 h according to the standard procedure described in the 

European Pharmacopoeia [24]. 

The oils were solubilized in n-hexane, dried over anhydrous sodium sulphate and dried under N

2

 to 


remove hexane. Samples were stored at +4 °C in the dark until tested and analyzed. 

3.3. GC-FID Analysis 

The GC-FID analysis  was carried out on a Perkin-Elmer Sigma-115 gas chromatograph equipped 

with a flame ionization detector (FID) and a data handling processor. The separation was achieved 

using an apolar HP-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). 

Column temperature: 40 °C, with 5 min initial hold, and then to 270 °C at 2 °C/min, 270 °C (20 min); 

injection mode: splitless (1 μL of a 1:1000 n-pentane solution). Injector and detector temperatures 

were 250 °C and 290 °C, respectively. Analysis was also run by using a fused silica HP Innowax 

polyethylenglycol capillary column (50 m × 0.20 mm i.d., 0.25 μm film thickness). In both cases, 

helium was used as carrier gas (1.0 mL/min). 

3.4. GC/MS Analysis 

The GC/MS analysis was performed on an Agilent 6,850 Ser. II apparatus, fitted with a fused silica 

DB-5 capillary column (30 m × 0.25 mm i.d., 0.33 μm film thickness), coupled to an Agilent Mass 

Selective Detector MSD 5973; ionization energy voltage 70 eV; electron multiplier voltage energy 

2000 V. Mass spectra were scanned in the range 40–500 amu, scan time 5 scans/s. Gas 

chromatographic conditions were as reported in the previous paragraph; transfer line temperature, 295 °C. 



3.5. Identification of the Essential Oils Components 

Most constituents were identified by gas chromatography by comparison of their Kovats retention 

indices (Ri) with either those of the literature [25,26] or with those of authentic compounds available 

in our laboratories. The Kovats retention indices were determined in relation to a homologous series of 



n-alkanes (C

10

–C



35

) under the same operating conditions. Further identification was made by 

comparison of their mass spectra on both columns with either those stored in NIST 02 and Wiley 275 


Int. J. Mol. Sci. 201213 16588 

 

 

libraries or with mass spectra from the literature [25,27] and a homemade library. The components 



relative concentrations were obtained by peak area normalization. No response factors were calculated.  

3.6. Biological Assay 

A bioassay based on germination and subsequent radicle growth was used to study the phytotoxic 

effects of the three essential oils on seeds of Raphanus sativus L. cv. “Saxa” (radish), Lepidium 

sativum  L. (garden cress) and the three weed species Sinapis arvensis L. (wild mustard), Triticum 

durum  L. (wheat) and Phalaris canariensis L. (canary grass). The seeds of radish and garden cress 

were purchased from Blumen srl (Piacenza, Italy), while mustard, wheat and canary grass were 

collected from wild plants. The seeds were surface sterilized in 95% ethanol for 15 s and sown in Petri 

dishes (Ø = 90 mm) containing five layers of Whatman filter paper impregnated with distilled water  

(7 mL, control) or a tested solution of the essential oil (7 mL) at the different assayed doses. The 

germination conditions were 20 ± 1 °C with a natural photoperiod. The essential oils, in a  

water–acetone mixture (99.5:0.5), were assayed at the doses of 2.5, 1.25, 0.625, 0.25, 0.125 and  

0.062 μg/mL. Controls performed with water-acetone mixture alone showed no appreciable differences 

in comparison with controls in water alone. Seed germination was observed directly in Petri dishes, 

each 24 h. A seed was considered germinated when the protrusion of the root became evident [28]. 

After 120 h (on the fifth day), the effects on radicle elongation were measured in centimeter. Each 

determination was repeated three times, using Petri dishes containing 10 seeds each. Data are 

expressed as the mean ± SD for both germination and radicle elongation. Data were ordered in 

homogeneous sets, and the Student’s test of independence was applied [29]. 



3.7. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal 

Concentration (MBC) 

The antibacterial activity was evaluated by determining the minimum inhibitory concentration 

(MIC) and the minimum bactericidal concentration (MBC) using the broth dilution method [30–32]. 

Eight bacterial species, selected as representative of the gram-positive and gram-negative classes, were 

tested: Staphylococcus aureus (ATTC 25923), Streptococcus faecalis (ATTC 29212), Bacillus subtilis 

(ATCC 6633), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), 



Staphylococcus epidermidis (ATCC 12228), Klebsiella pneumoniae (ATCC 10031) and Proteus 

vulgaris (ATCC 13315). The strains were maintained on Tryptone Soya agar (Oxoid, Milan, Italy); for 

the antimicrobial tests, Tryptone Soya broth (Oxoid, Milan, Italy) was used. In order to facilitate the 

dispersion of the oil in the aqueous nutrient medium, it was diluted with Tween 20 at a concentration 

of 10%. Each strain was tested with a sample that was serially diluted in broth to obtain concentrations 

ranging from 100 μg/mL to 0.8 μg/mL. The sample was previously sterilized with a 0.20 μm Millipore 

filter. The sample was stirred, inoculated with 50 μL of physiological solution containing 5 × 10

6

 

microbial cells and incubated for 24 h at 37 °C. The MIC value was determined as the lowest 



concentration of the sample that did not permit any visible growth of the tested microorganism after 

incubation. The control, containing only Tween 20 instead of the essential oil, was not toxic to the 

microorganisms. Cultures, containing only sterile physiologic solution Tris buffer, were used as 

positive control. MBC was determined by subculture of the tubes with inhibition in 5 mL of sterile 



Int. J. Mol. Sci. 201213 16589 

 

 

nutrient broth. After incubation at 37 °C, the tubes were observed. When the germs did not grow, the 



sample denoted a bactericidal action. Oil samples were tested in triplicate and the experiment was 

performed three times. The results are expressed as mean ± SD. Chloramphenicol was used as 

reference drug [17].  

4. Conclusions  

Data obtained in this paper could be useful in the chemotaxonomic knowledge of the genus 



Melaleuca that consists of about 230 species, the chemistry of M. armillaris,  M.  styphelioides  and  

M. acuminata being little known. Data on phytotoxic activities could help to explain the ecological 

role of genus Melaleuca in the Mediterranean area. The antimicrobial activity of the essential oil is in 

agreement with the uses of other Melaleuca species as antimicronial drugs. 

Conflict of Interest 

The authors declare no conflict of interest. 



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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article 

distributed under the terms and conditions of the Creative Commons Attribution license 



(http://creativecommons.org/licenses/by/3.0/). 

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