[P-069]
ESSENTIAL OIL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF
Phlomis lanata ENDEMIC IN GREECE

Andromachi TANIMANIDIS, Maria COULADIS, Olga TZAKOU,
Ioanna B. CHINOU and Catherine HARVALA
Department of Pharmacognosy, School of Pharmacy, University of Athens,
Panepistimioupolis Zografou 15771, Greece

ABSTRACT

The essential oil of Phlomis lanata has been analyzed by GC/MS. 48 compounds (96.85%) of the oil were identified; a-pinene, limonene and trans-caryophyllene were found as main components. The essential oil showed a moderate activity against six Gram (+) bacteria and a stronger against the three tested pathogenic fungi.


INTRODUCTION

Phlomis L. (Labiatae) comprises 12 taxa naturalized in Europe and its species are mentioned by Dioskorid as herbal drugs. P. lanata is a shrub, endemic of Crete.


MATERIALS AND METHODS

Aerial parts of P. lanata were collected in Crete, Greece. A voucher specimen has been deposited in the Laboratory of Pharmacognosy, University of Athens.

Plant material was subjected to hydrodistillation using a modified Clevenger-type apparatus. Then, the oil was submitted to GC/MS. The GC-MS analyses were carried out using a Hewlett Packard 6890/5973 system, with a HP-5MS capillary column (30m x 0.25 mm; film thickness: 0.25 mm) GC oven initial temperature was 60°C and programmed to 280°C at a rate of 3°C/min. The identification of the chemical constituents was based on comparison of their retention Kovats indices, their retention times and mass spectra with those obtained from authentic samples and/or the NBS/NIST and the literature.

Antimicrobial activity

The bacteriostatic activity of the essential oil against the two Gram (+) bacteria: Staphylococcus aureus, S. epidermidis the four Gram (-) bacteria: E., Ent. cloacae, Kl. pneumoniae and Ps. aeruginosa and the antifungal activities against the pathogens fungi C. albicans, C. tropicalis and T. glabrata were determined, using the dilution technique, the MIC was measured for the oil, a-pinene and limonene (Table 2). Standard antibiotics (netilmicin, and amoxicillin with clavulanic acid) were used in order to control the sensitivity of the tested bacteria and 5-flucytocine, amphotericin B and intraconazole were used in order to control the tested fungi.


RESULTS

Results obtained in the qualitative and quantitative analysis are shown in Table 1. Fourty eight constituents were identified and quantified from the oil of P. lanata representing 98.85% (area percent) of the total oil. The major constituents were a-pinene (25.41%), limonene (15.67%), and trans-carryophyllene (8.76%).

The results on the bioassays showed that the oil appeared a moderate activity against the four of the six Gram (+) tested bacteria and a stronger one against the Gram (-) bacteria E. coli and P. aeruginosa (MIC values 3-6.5).


Table 1. Main components (%) of P. lanata
    Constituentsa
%
KIb
    Constituentsa
%
KIb
1
a-Thujene
tr
926
25
Isocomene
4.91
1377
2
a-Pinene
25.41
936
26
b-Cubebene
tr
1380
3
Sabinene
0.65
967
27
b-Elemene
tr
1381
4
b-Pinene
1.74
972
28
(E)-Caryophyllene
8.76
1413
5
b-Myrcene
1.58
985
29
b-Gurjunene
tr
1418
6
a-Phellandrene
1.27
1000
30
a-Himachalene
1.80
1441
7
a-Terpinene
0.87
1014
31
a-Humulene
tr
1443
8
p-Cymene 
tr
1023
32
Dehydroaromadendrene
3.18
1445
9
Limonene
15.67
1027
33
9-epi-(E)-Caryophyllene
tr
1456
10
(Z)-b-Ocimene 
2.89
1032
34
g-Muurolene
4.53
1473
11
Benzene acetaldehyde
tr
1036
35
(E)-b-Ionone 
0.33
1476
12
(E)-b-Ocimene 
2.07
1041
36
cis-b-Guaiene 
0.49
1480
13
g-Terpinene
0.42
1051
37
Valencene
tr
1482
14
a-Terpinolene
1.32
1081
38
d-Cadinene
1.51
1513
15
Linalool
0.78
1093
39
a-Calacorene
tr
1530
16
Nonanal
1.03
1098
40
(E)-Nerolidol
0.68
1553
17
a-Campholenal
tr
1120
41
Caryophyllene oxide
2.86
1571
18
Terpin-4-ol
tr
1167
42
Humulene epoxide II
0.35
1597
19
a-Terpineol
0.24
1182
43
Tetradecanal
tr
1600
20
Decanal
0.26
1197
44
Hexadecanal
0.77
1705
21
Geraniol
tr
1246
45
Trimethylpentadecanonec
0.94
1840
22
Carvacrol
3.26
1295
46
Hexadecanoic acid
0.85
1963
23
Cyclosativene
0.34
1357
47
Tetramethylhexadecenold
3.54
2108
24
a-Copaene
1.26
1366
48
Tricosane
0.29
2310
               Total
96.85
  
aCompounds listed in order of elution from a HP-5 MS column.
bKovats Indices (KI) on HP-5 MS.
c6,10,14-trimethyl-2-Pentadecanone
d3,7,11,15-tetramethyl-2-Hexadecen-1-ol

Table 2. Antimicrobial activity (MIC mg/ml) of P. lanata and its main components
Ess. oil
1
2
3
4
5
6
7
8
9
P. lanata
> 20
> 20
6.50
> 20
8
3
4.95
4.80
4.65
a-pinene
7.5
9.5
6
15
8
2
4
4
2
limonene
-
-
-
-
-
-
-
-
-
Incz
-
-
-
-
-
-
1·10-3
0.1·10-3
1·10-3
5-Flct
-
-
-
-
-
-
0.1·10-3
1·10-3
10·10-3
Amp- B
-
-
-
-
-
-
1·10-3
0.5·10-3
4·10-4
NET
4·10-3
4·10-3
8.8·10-3
8·10-3
8·10-3
10·10-3
-
-
-
AMC
2·10-3
2·10-3
2.4·10-3
2.8·10-3
2.2·10-3
2·10-3
-
-
-
Clav. acid
0.5·10-3
0.5·10-3
1·10-3
1.6·10-3
1·10-3
1.2·10-3
-
-
-
1. Staphylococcus aureus, 2. S. epidermidis, 3. P. aeruginosa, 4. E. cloacae, 5. Klebsiella pneumoniae, 6. Escherichia coli, 7. Candida albicans, 8. C. tropicalis, 9. Torulopsis glabrata; AMC (=amoxicillin), NET (=Netilmicin), AMC (=Amoxicillin), Clav. acid (=Clavulanic acid), Incz (=Intraconazole), 5-Flctc (=5-flucytocine), Amph-B (Amphotericin B)

DISCUSSION

It is noteworthy that the oil exhibited a specific activity against all the three tested pathogenic fungi (MIC values 4.65-4.95mg/ml). In the screening standard of the pure monoterpene a-pinene as well as limonene, which were found to be the major compounds of the oil (25.4% and 15.67% respectively), were tested on the same cultures under identical conditions to compare their activity with that of the investigated oil. The results suggest that the activity of the oil can be attributed, to a considerable degree, to the existence of a-pinene which appears to possess a similar activity against all the tested microorganisms, as limonene appeared completely inactive.


LITERATURE
  1. De Filipps R.A. (1972): In: Flora Europaea, Tutin T.G, Heywood V.H, Burges N.A, Moore D.M, Valentine S.M, Webb D.A, editors, Cambridge University Press Vol.3., Cambridge, 145.

  2. Gennadios P.G. (1997): Phytological Dictionary, Trohalia ed, Athens, 999.

  3. Janssen A.M, Scheffer J.J.C., Baerheim Svendsen A.(1987): Antimicrobial Activity of Essential Oils: A 1976-1986 Literature Review. Aspects of the Test Methods, Planta Medica. 5, 395-397.

[P-069]