2007, 12, 772-781 molecules issn 1420-3049




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Molecules 2007, 12, 772-781

molecules

ISSN 1420-3049

© 2007 by MDPI


www.mdpi.org/molecules
Full Paper
Chemical Composition and Antimicrobial Activity of the Essential Oil of Algerian Phlomis bovei De Noé subsp. bovei
Christos Liolios 1, Hocine Laouer 2, Nacira Boulaacheb 2, Olga Gortzi 3 and Ioanna Chinou 1,*
1 Department of Pharmacy, Division of Pharmacognosy, Chemistry of Natural Products, University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece; E-mail: liolios@pharm.uoa.gr;
Fax: (+30) 2107274115.

2 Department of Biology, Faculty of Sciences, University Ferhat Abbas, Setif, Algeria;
E-mails: hocine_laouer@yahoo.fr; boulaacheb1bv@yahoo.fr; Fax: (+213) 36 92 51 22

3 Department of Food Technology, Technological Educational Institution (T.E.I.) of Larissa, Terma Temponera str., Karditsa, Greece; E-mail: ogkortzi@teiath.gr; Fax: (+30) 2441072070
* Author to whom correspondence should be addressed; e-mail: ichinou@pharm.uoa.gr,
Fax: (+30) 2107274115
Received: 27 February 2007; in revised form: 27 March 2007 / Accepted: 3 April 2007 / Published:
12 April 2007

Abstract: The chemical composition of essential oil obtained by steam distillation of dried aerial parts of Phlomis bovei De Noé subsp. bovei collected from Algeria, was analyzed by GC and GC/MS. Seventy five constituents (corresponding to 86.37% of the total weight) were identified. The main components were: germacrene D, β-caryophyllene, β-bournonene, thymol and hexahydrofarnesyl acetone. Furthermore, the antimicrobial activity of the oil was evaluated against six Gram (+/-) bacteria and three pathogenic fungi, using the agar dilution technique. It was found that the oil exhibited strong antimicrobial activity against most of the tested microorganisms.


Keywords: Phlomis bovei De Noé; chemical composition; essential oil; antimicrobial activity.

Introduction

The plants of the genus Phlomis are native to Turkey, North Africa, Europe and Asia. Phlomis bovei De Noé, syn. Phlomis samia Desfontaines (Lamiaceae) is a rare Algerian endemic plant, commonly known as Kayat El Adjarah [1] in the Algerian dialect or variously named Farseouan, Tarseouan, Iniji, R’ilef and Azaref throughout the North of Africa [2]. It is one among the nine endemic plants recorded in the ‘Rapport National sur la Diversité Biologique’ [1]. P. bovei is a herbaceous perennial plant, which grows up to 0.8 m. and often develops a stout woody base. All parts are sticky, because of its dendroid stellate glandular hairs. Its basal leaves are green, heart-shaped, with scalloped margins, 6.5­25 x 4.5­20 cm and it has a petiole of between 4­18 cm in length. To date two subspecies have been recorded for P. bovei De Noé: P. bovei De Noé subsp. bovei and P. bovei De Noé subsp. maroccana Maire. The present study refers to the former, which to our knowledge has never been studied phytochemically before, whereas previous studies on the essentials oils of Phlomis species from around the Mediterranean have included: Phlomis fruticosa, P. cretica, P. samia, P. lanata, P. linearis, P. leucophracta, P. chimerae and P. grandiflora var. grandiflora.


Results and Discussion
The essential oil obtained by hydrodistillation of aerial parts of Phlomis bovei De Noé subsp. bovei was light yellow in color and possessed a distinct sharp odor. The yields were 0.22 % w/w. The analysis of the volatile constituents was carried out using two different GC-MS systems, equipped with two columns of different polarities (HP-5 and Aquawax, respectively). The chemical compositions are summarized in Tables 1 and 2. The identified components represented 86.37% of all the components found in the oil samples. These percentages were based on normalization of peak areas without application of the response correction factor. The major components included: germacrene D (21.45%), thymol (8.43%), β-caryophyllene (7.05%) and hexahydrofarnesyl acetone (5.84%). We should also note the presence in the essential oil of a total 6.03 % of normal saturated hydrocarbons (see Table 2). Although most of the identified constituents occurred in both methods of analysis, it was also noted that some chemical constituents occurring in appreciable amounts in HP-5 were absent in Aquawax and viceversa. This was due to the differences between the GC-MS instruments, the two columns and the absence of reference retention indexes for the second column. Thus the identification of the components for the second column was based on their mass spectra and by comparison of their retention times with those of authentic samples.
Table 1. Main components of the essential oil from the aerial parts of P. bovei De Noé.




Compounds*

RI§

% in Essent. oil

Method of identification

HP-5

Aquawax



1-Octen-3-ol

977

1505

1.08

a, b, d,



3-Octanol

993

1394

0.07

a, b, d



n-Octanal

1004

1289

0.08

a, b, d



E,E-2,4-Heptadienal

1012

-

0.03

a, b, d



p-Cymene

1023

1270

0.04

a, b, c, d


Table 1. Cont.



Limonene

1024

-

0.05

a, b, c,d



Phenylacetaldehyde

1046

-

0.02

a, b, d



γ-Terpinene

1058

1246

0.06

a, b, d



n-Octanol

1071

1574

0.13

a, b, d



Linalool

1104

1570

0.43

a, b, d



Nonanal

1107

-

0.8

a, b, d



Benzeneethanol

1115

1896

0.06

a, b, d



trans-2-Nonenal

1160

1555

0.29

a, b, d



Terpine-4-ol

1175

1593

0.21

a, b, d



α- Terpineol

1187

-

0.24

a, b, c, d



Caprylic acid [Octanoic acid]

1189

2021

0.12

a, b, d



Methyl salicylate

1191

1749

0.05

a, b, d



n-Decanal

1203

-

0.15

a, b, d



β-Cyclocitral

1215

-

0.04

a, b, d



Thymol methyl ether

1225

-

0.04

a, b, d



Carvacrol methyl ether

1230

-

0.04

a, b, d



trans-2-Decenal

1269

1630

0.32

a, b, d



Thymol

1296

2065

8.43

a, b, d, c



Carvacrol

1303

-

1.03

a, b, d, c



trans,trans-2,4-Decadienal

1328

1783

0.15

a, b, d



Thymol methyl ester

1331

-

0.05

a, b, d



α-Cubebene

1342

-

0.17

a, b, d



2-Undecenal

1359

-

0.54

a, b, d



α-Copaene

1364

1523

0.73

a, b, d



β-Bourbonene

1376

1540

2.96

a, b, d, c



trans-β-Damascenone

1387

1789

0.68

a, b, d



β-Elemene

1389

-

1.42

a, b, d



Dodecanal

1399

-

0.31

a, b, d



β-Caryophyllene

1418

1586

7.05

a, b, d, c



trans -β-Copaene

1423

-

0.66

a, b, d



β-Gurjunene

1432

-

0.41

a, b, d



α-Humulene

1443

1646

1.45

a, b, d



trans-β-Farnesene

1462

1664

1.49

a, b, d,



Germacrene D

1475

1689

21.45

a, b, d, c



α-Selinene

1499

-

0.83

a, b, d,



α-Muurolene

1503

1763

0.38

a, b, d



Germacrene A

1509

-

0.27

a, b, d



β-Bisabolene

1510

1713

1.08

a, b, d



Butylated hydroxytoluene [Ional]

1515

1905

0.88

a, b, d



epi-Bicyclosesquiphellandrene

1517

1582

0.03

a, b, d



δ-Cadinene

1525

1736

2.16

a, b, d, c


Table 1. Cont.



Cadina-1(2),4-dien

1532

-

0.34

a, b, d



α- Cadinene

1535

-

0.22

a, b, d



α- Calacorene

1540

-

0.16

a, b, d



Nerolidol

1561

2014

0.36

a, b, c, d



Spathulenol

1577

2037

0.79

a, b, d



Caryophyllene Oxide

1584

1947

2.41

a, b, d, c



Copaen-4-Α-Ol

1585

-

0.43

a, b, d



nor-Copaenone

1627

-

0.11

a, b, d



Cadina-1,4-Dien-3-Ol

1628

-

0.19

a, b, d



epi-α-Muurolol

1643

-

0.97

a, b, d



α-Muurolol [Torreyol]

1649

-

0.7

a, b, d



Amylcinnamalaldehyde

1662

2081

0.54

a, b, d



α-Cadinol

1664

-

2.38

a, b, d



Eudesmadienol derivative

1685

-

2.56

a, b, d



Hexahydrofarnesyl acetone (6,10,14-Trimethyl-2-pentadecanone)

1851

2043

5.84

a, b, d



Nonadecane

1900

1900

0.19

a, b, d



Farnesyl acetone B

1925

-

0.24

a, b, d



Hexadecanoic acid methyl ester

1926

-

0.14

a, b, d



Eicosane

2000

2000

0.19

a, b, d



Heneicosane

2100

2100

0.21

a, b, d



Docosane

2200

2200

0.18

a, b, d



Tricosane

2300

2300

1.4

a, b, d



Tetracosane

2400

2400

0.27

a, b, d



Pentacosane

2500

2500

1.21

a, b, d



Hexacosane

2600

2600

0.06

a, c, d



Heptacosane

2700

2700

1.19

a, c, d



Octacosane

2800

2800

0.13

a, c, d



Triacontane

3000

3000

0.66

a, c, d



Hentriacontane

3100

3100

0.34

a, c, d

Total:

86.37




*Compounds listed in order of elution from a HP-5 MS column.

§Retention indices (KI) on HP-5 MS capillary column.

a= Retention time; b = Retention Index; c = Peak enrichment; d = mass spectra.


Table 2. Composition of P. bovei De Noé subsp. bovei essential oil by substance class.

Compounds

% in essential oil

Monoterpenes

Sesquiterpenes

Saturated


0.15

43.26

6.03

Hydrocarbons total :

49.44

Alcohols

Aldehydes

Ketones, Ethers, Acids, Esters, Oxides


18.38

3.27

12.28

Oxygenated compounds total:

36.93

Total compounds:

86.37

For the essential oil obtained from the leaves of P. fruticosa collected in Montenegro (Table 3) the main constituents were: β-caryophyllene (12.0%), (E)-methyl-isoeugenol (15.3%), α-asarone (10.9%), caryophyllene oxide (8.1%) and α-pinene (6.6%)[3]. The antimutagenic activity of the essential oil and of the crude extract was evaluated by the same research group [4]. Studies on the same plant, from the same region, have been conducted considering the antimicrobial and the antifungal activity of its essential oil, as well as its methanolic extract, with moderate results [5]. Traditionally the infusion of P. fruticosa leaves is used in Greece as a tonic drink, whereas in Italy the dried leaves are used as a poultice on wounds [6].

The flowers of P. fruticosa collected in Greece (Table 3) yielded an essential oil rich in germacrene D (17.8%), γ-bisabolene (12.6%), α-pinene (8.9%) and β-caryophyllene (8.7%) [7]. In another study on the essential oil from the aerial parts of P. fruticosa collected in central-East Peloponnesus, the main constituents were: germacrene D (21.4%), Z-γ-bisabolene (7.1%), α-pinene (12.6%) and β-caryophyllene (12.6%) and linalool (8.0%) [8]. In the same study, the volatile constituents of two other Greek Phlomis species - P. cretica and P. samia - were studied. For P. cretica the major compounds were: α-pinene (9.4%), limonene (7.1%), cis-β-ocimene (5.4%), linalool (7.5%), β-caryophyllene (17.3%) and germacrene D (20.1%). P. samia also exhibited large amounts of β-caryophyllene (5.8%), germacrene D (6.3%) and linalool (2.3%) but its major compound was (E)-β-farnesene (20.7%). The essential oils were tested against Gram (±) bacteria and fungi, showing moderate activity [8].

The main chemicals identified in the essential oil of the aerial parts of P. lanata, another Phlomis growing in Greece (Table 3) were: α-pinene (25.41%), limonene (15.67%), β-caryophyllene (8.76%), isocomene (4.91%) and γ-muurolene (4.53%). The essential oil of the plant was tested against Gram (±) bacteria and fungi. Like the previous study, it showed moderate antimicrobial activity, with the exception of E. coli and P.aeruginosa, towards which it exhibited stronger activity [9].



P. linearis Boiss. & Bal., growing in central East and Southeast Anatolia, an endemic Phlomis of Turkey, was characterized by the predominance of: β-caryophyllene (24.2%), germacrene D (22.3%) and caryophyllene oxide (9.2%) [10].

Table 3. Main components of the essential oils from different Mediterranean Phlomis species.

Components

Phlomis bovei De Noé subsp. bovei

P.cretica [8]

P.fruticosa [3]


P.fruticosa [7]


P.fruticosa [8]


P.samia [8]

P.linearis [10]

P.lanata [9]

P.leucophracta [11]

P.cimereae [11]

P.grandiflora var.grandiflora [11]

Hexahydro-Farnesyl Acetone

5.84

-

-

-

-

-

-

-

-

0.40

-

Spathulenol

0.79

0.10

0.50

-

-

3.70

-

-

0.30

-

0.40

α -Pinene

-

9.40

6.60

8.90

12.60

0.80

-

25.41

19.20

11.00

2.40

Limonene

0.05

7.10

0.50

0.40

0.90

0.10

-

15.67

11.00

5.50

2.70

cis-β-Ocimene

-

5.40

-

-

0.50

-

-

2.89

-

0.40

0.60

δ-Cadinene

2.16

1.20

0.90

1.80

1.00

2.40

1.00

1.51

0.40

5.00

1.30

(E)-Methyl-Isoeugenol

-

-

15.30

-

-

-

-

-

-

-

-

γ-Bisabolene

1.08

-

1.40

12.60

7.10

-

-

-

-

0.20

2.50

α-Asarone

-

-

10.90

-

-

-

-

-

-

-

-

Thymol

8.43

-

0.20

-

-

-

0.50

-

-

-

-

Germacrene D

21.45

20.10

2.30

17.80

21.40

6.30

22.30

-

4.50

6.10

45.40

β-Caryophyllene

7.05

17.30

12.00

8.70

12.60

5.80

24.20

8.76

20.20

31.60

22.80

γ-Muurolene

-

-

-

-

-

-

0.40

4.53

tr

-

tr

Linalool

-

7.50

0.60

0.70

8.00

2.30

0.60

0.78

-

4.70

0.60

E-β-Farnesene

1.49

-







0.60

20.70

-

-

1.10

0.50

1.00

Caryophyllene Oxide

2.41

0.60

8.10

1.90

0.80

3.20

9.20

2.86

1.70

4.80

0.40

Bicyclogermacrene

-

-

-

-

-

-

1.10

-

0.80

-

4.90

The essential oils of three other Turkish Phlomis species (Table 3) have also been studied previously [11]. The essential oil of P. leucophracta consisted mainly of β-caryophyllene (20.2%), α-pinene (19.2%) and limonene (11.0%), while in P. chimerae the principal compounds were β-caryophyllene (31.6%), α-pinene (11.0%), germacrene D (6.1%), limonene (5.5%) and linalool (4.7%), and in P. grandiflora var. grandiflora: germacrene D (45.4%), β-caryophyllene (22.8%) and bicyclogermacrene (4.9%) have been identified among the most abundant constituents [11]. The oils of P. bovei De Noé and of the other Mediterranean species: P. grandiflora var. grandiflora [11], P. cretica [8], P. fruticosa [3, 7, 8], P. samia [8], P. linearis [10], P. lanata [9], P. leucophracta [11] and P. cimereae [11], presented great amounts of the sesquiterpenoids germacrene D, E-β-farnesene and β-caryophyllene. In accordance to these results, in our study besides the presence of germacrene D (21.45%) and β-caryophyllene (7.05%), hexahydrofarnesyl acetone (5.84%) has been also identified among the most abundant compounds, which could be considered as the biosynthetic predecessor of the above referred sesquiterpenoids, from the well known mevalonic acid pathway [12].

The essential oil of Phlomis bovei De Noé subsp. bovei exhibited a wide profile of antimicrobial activity against most of the tested microorganisms, in comparison with the tested antibiotics and the standards β-caryophyllene and thymol (Table 4), while only K. pneumoniae appeared to be a microorganism displaying significant resistence. Considering the fact that β-caryophyllene possesses in general moderate antimicrobial activity, we conclude that the antimicrobial activity of the essential oil from P. bovei can be attributed, to a considerable degree, to the presence of germacrene D and thymol, which are well known to posses strong antimicrobial activity [13-15].
Table 4. Antimicrobial activities (MIC mg/mL) of the studied Phlomis essential oils and its main components.

Species-Essential Oils

S. aureus

S. epidermidis

P. aeruginosa

E. cloacae

K. pneumoniae

E. coli

C. albicans

C. tropicalis

C. glabrata

P. bovei

0.98

±0.004


0.85

±0.007


1.00

±0.011


1.37

±0.018


4.75

±0.035


1.12

±0.018


1.35

±0.008


0.95

±0.015


0.89

±0.013


β-Caryophyllene

>20

>20

>20

>20

>20

>20

-

-

-

Thymol

1.25

±0.010


1.38

±0.008


2.45

±0.022


2.00

±0.005


2.88

±0.027


1.70

±0.023


1.50

±0.013


1.34

±0.020


1.18

±0.018


Itraconazole

-

-

-

-

-

-

1x10-3

0.1x10-3

1x10-3

5-Flucytocine

-

-

-

-

-

-

0.1x10-3

1x10-3

10x10-3

Amphotericin B

-

-

-

-

-

-

1x10-3

0.5x10-3

0.4x10-3

Netilmicin

4x10-3

4x10-3

8.8x10-3

8x10-3

8x10-3

10x10-3

-

-

-

Amoxicillin

2x10-3

2x10-3

2.4x10-3

2.8x10-3

2.2x10-3

2x10-3

-

-

-

Clavulanic acid

0.5x10-3

0.5x10-3

1x10-3

1.6x10-3

1x10-3

1.2x10-3

-

-

-


  • = not active

Conclusions
Our GC and GC/MS study of the essential oil from Algerian Phlomis bovei De Noé led to the identification of 75 constituents (corresponding to 86.37% of the total weight) among which germacrene D, β-caryophyllene, β-bournonene, thymol, and hexahydrofarnesyl acetone were the main ones. The oil exhibited a broad spectrum of strong antimicrobial activities and it possessed a much better antimicrobial activity in comparison with all previously tested and assayed samples from Greek Phlomis species [8], showing that this plant oil could have a commercial potential as an antiseptic agent, however, further investigation should be carried out against new series of pathogenic microorganisms.
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