Open Access

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia

  • Zied Zarai1,
  • Adel Kadri2Email author,
  • Ines Ben Chobba3,
  • Riadh Ben Mansour4,
  • Ahmed Bekir5,
  • Hafedh Mejdoub6 and
  • Néji Gharsallah3
Lipids in Health and Disease201110:161

https://doi.org/10.1186/1476-511X-10-161

Received: 23 August 2011

Accepted: 21 September 2011

Published: 21 September 2011

Abstract

Background

In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.

Methods

The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test.

Results

The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 μg/ml.

Conclusion

This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.

Keywords

Antimicrobial cytotoxicity essential oil Marrubium vulgare L., pathogenic microorganisms HeLa cell lines

Background

The Lamiaceae plants was considered as one of the large plant families used as a framework to evaluate the occurrence of typical secondary metabolites [1]. The genus Marrubium comprises 10 species, which are found wild in many regions of Tunisia. Among them, Marrubium vulgare L. is a perennial herb of the Labiatae family which is commonly known as "horehound" in Europe, or "Marrubia" in Tunisia, is naturalized in North and South America, the latter and Western Asia. It possesses tonic, aromatic, stimulant, expectorant, diaphoretic and diuretic properties. It is helpful for bronchial asthma and non-productive cough. It was formerly much esteemed in various uterine, visceral and hepatic affections and in phthisis [2]. In the Mediterranean region, M. vulgare is frequently used in folk medicine to cure a variety of diseases. The plant is reported to possess hypoglycemic [3], vasorelaxant [4], antihypertensive [5], analgesic [6, 7], anti-inflammatory [8], antioxidant activity [9, 10], antiedematogenic activity [11], and many other biological activities. In Tunisian folk medicine, it was used as hypotensive, hypoglycemic and cardiotonic.

Recently, a large number of essential (volatile) oils and their constituents have been investigated for their biological activity, notably antibacterial, antifungal, and antioxidant properties [1214]. Essential oils and their components are gaining increasing interest as a natural alternative to synthetic drugs [15], particularly against microbial agents because of their relatively safe status wide acceptance by consumers and their exploitation for potential multipurpose functional use. The chemical compositions of M. vulgare essential oil from various origins have been the subject of many studies. The literature reveals the occurence of several chemotypes. From Lithuania, (Z)-β-farnesene, β-caryophyllene, (E)-2-hexenal, α-humulene and germacrene D were the main components of M. vulgare essential oil [16]. From Czech Republic, the main constituents of M. vulgare essential oil were β-caryophyllene and germacrene D [17]. From different region of Iran, the main constituants of M. vulgare essential tricyclene, β-pinene, bisabolol, β-elemone and isomenthon-8-thiol [18], β-bisabolene, 8-cadinene and isocaryophyllene [19], and bisabolene, β-caryophyllene, germacrene D and E-β-farnesene [20], caryophyllene oxide, β-caryophyllene and germacrene D [21].

The interest in plants with antimicrobial properties has been revived because of current problems associated with the use of antibiotics [22]. Therefore, essential oils and other naturally occurring antimicrobials are attractive to the food industry as well as imparting flavor [23]. More recently, the essential oil of this plant was advocated for their use as an antioxidant agent [10], but to the best of our knowledge, there are no reports on the antimicrobial properties and the cytotoxicity has been published.

Therefore, this paper was conducted to investigate for the first time the antimicrobial properties against clinical and pathogenic microorganisms and the cytotoxicity of M. vulgare essential oil grown in Tunisia.

Methods

Chemicals, reagents and plant material

Chemicals and reagents were supported by Prolabo (Paris, France) and Pharmacia (Uppsala, Swedeen). Plant materials (aerial parts) of M. vulgare L. were grown in the vicinity of the village of Ouled Mnasser, with a latitude of 34.88 (34° 52' 60 N) and a longitude of 9.13 (9° 7' 60 E) in Sidi Bouzid, Tunisia. The aerial parts of wild growing plant have been collected during the period of June-July 2009. The plant materials were confirmed by a senior A. bekir. Voucher specimens were deposited at ISET, Sfax (Département de Génie des procédés) as Bekir 520.

Distillation of essential oil and GC/MS analysis conditions

The fresh aerial parts of M. vulgare (300 g) were hydrodistilled using a Clevenger-type apparatus to recover the essential oils for 4 h. The distilled essential oils were dried over anhydrous sodium sulfate, filtered and stored at +4°C.

The essential oil was analyzed using an Agilent-Technologies 6890 N Network GC system equipped with a flame ionization detector and HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm; Agilent-Technologies, Little Falls, CA, USA). The injector and detector temperatures were set at 250 and 280°C, respectively. The column temperature was programmed from 35 to 250°C at a rate of 5°C/min, with the lower and upper temperatures being held for 3 and 10 min, respectively. The flow rate of the carrier gas (helium) was 1.0 ml/min. A sample of 1.0 μl was injected, using split mode (split ratio, 1:100). All quantifications were carried out using a built-in data-handling program provided by the manufacturer of the gas chromatograph. The composition was reported as a relative percentage of the total peak area. The identification of the essential oil constituents was based on a comparison of their retention times to n-alkanes, compared to published data and spectra of authentic compounds. Compounds were further identified and authenticated using their mass spectra compared to the Wiley version 7.0 library.

Antimicrobial activity assay

Microbial strain

The essential oil of M. vulgare was individually tested against a panel of microorganisms (Table 1). The antimicrobial activities of essential oil were determined against sixteen of human-pathogenic microbial strains. The bacteria and fungi used were selected because they have implicated with skin, oral and intestinal tract of man. Twelve species of bacteria and four species of fungi as shown in Table 1 were used in this study.
Table 1

Pathogenic bacteria and fungi used for the antimicrobial assay.

Bacteria

 

   Staphylococcus aureus 1327

Staphylococcus epidermidis

   Micrococcus luteus

Enterococcus faecalis

   Enterobacter cloacae

Staphylococcus aureus 25923

   Bacillus subtilis

Bacillus cereus

   Pseudomonas aeruginosa 27853

Klebsielle pneumoniae WHO24

   Escherchia coli 25922

 

Fungi

 

   Botrytis cinerea

Fusarium solan

   Penicillium digitatum

Aspergillus niger

Agar diffusion method

The agar diffusion method was employed for the determination of antibacterial activities of M. vulgare essential oil according to the method described by Berghe and Vlietinck (1991) [24]. The essential oil extracts were dissolved in 100% ethanol to a final concentration of 10 mg/ml and sterilized by filtration trough 0.22 μm Nylon membrane filter. The bacterial strains were cultured in a nutriment broth for 24 hours. Then, 200 μl of each suspension bacteria (106 CFU estimated by absorbance at 600 nm) was spread on Luria Broth agar. Bores were made by using a sterile borer and were loaded with 10 μl of each sample extract. Ethanol was used as negative control and ampicillin (10 μg/puit) as positive reference standard. All the plates were incubated at 37°C for 24 hours. Antibacterial activity was evaluated by measuring the zone of inhibition in millimetres. All experiments were done in triplicates.

Determination of the minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) values, which represent the lowest essential oil concentration that completely inhibits the growth of microorganisms, were determined by a micro-well dilution method [25]. The inoculum of each bacterium was prepared and the suspensions were adjusted to 106 CFU/ml. All the extracts were dissolved in 100% ethanol and then dilutions series were prepared in a 96-well plate. Each well of the microplate included 40 μl of the growth medium, 10 μl of inoculums and 50 μl of the diluted sample extract. The ampicillin and ethanol are used as positive and negative controls, respectively. The plates were then covered with the sterile plate and incubated at 37°C for 24 h. After that, 40 μl of 3- (4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) at a final concentration 0.5 mg/ml freshly prepared in water was added to each well and incubated for 30 min. The change to red colour indicated that the bacteria were biologically active. The MIC was taken to the well, where no change of colour of MTT was observed. The MIC values were done in triplicate.

Antibacterial assay disc-diffusion method

An antibacterial activity of the essential oils was screened against eight human pathogenic bacteria. The inhibitory effect on bacterial growth was determined using agar-disc diffusion assay [26, 27]. The bacterial cultures were first grown on Muller Hinton agar (MH) plates at 37°C for 18 to 24 h prior to seeding onto the nutrient agar. One or several colonies of the respective bacteria were transferred into API suspension medium (bioMerieux) and adjusted to 0.5 McFarland turbidity standards with a Densimat (bioMerieux) [28, 29]. The inocula of the respective bacteria were streaked into MH agar plates using a sterile swab and were then dried at 37°C during 15 min. A sterile filter disc having 6 mm of diameter was placed at the surface of MH agar and 5 μl of the essential oil was dropped onto each Whatman paper disc [30]. The treated Petri dishes were incubated at 37°C for 18 to 24 h. The antibacterial activity was evaluated by measuring the clear zone surrounding the Whatman paper. Standard discs of the antibiotic ampicillin were applied as a positive antibacterial controls.

Antibacterial assay dilution method

The minimal inhibitory concentration (MIC) of essential oil was determined using the Mueller Hinton broth (MHB) dilution method [31]. All tests were performed in MHB supplemented with ethanol 5% [32]. Bacterial strains were cultured overnight in MHB at 37°C. Tubes of MHB containing various concentrations of oils were inoculated with 10 μl bacterial inoculums adjusted to 106 CFU/ml. They were incubated under shaking conditions (100-120 rpm) at 37°C for 24 h [33, 34]. Control tubes without tested samples were essayed simultaneously. The essays were performed in triplicate. The MIC was defined as the lowest concentration preventing visible growth [35, 36].

Antifungal assay disc-diffusion method

The biological activity against yeasts was determined by employing disc agar diffusion method using Sabouraud Dextrose agar [37]. An aliquot (5 μl) of the oil was deposited on sterile paper discs (6 mm diameter) which were subsequently placed in the centre of the inoculated Petri dishes. After an incubation period of the 24 h at 30°C, the inhibitory activity was compared to that of commercial cycloheximide at a concentration of 10 mg/ml.

Cell lines and culture condition

HeLa cells (cervical cancer line, adherent) were used to investigate the cytotoxicity effect of essential oil. This cell line were grown in RPMI 1640 medium (Gibco) supplemented with 10% (v/v) foetal calf serum (FCS) and 2 mM L-glutamin in tissue culture flasks (Nunc). They were passed twice a week and kept at 37°C in a humidified atmosphere of 95% air and 5% CO2.

MTT test

The proliferation rates of HeLa cells after treatment with essential oils were determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The yellow compound MTT is reduced by mitochondrial dehydrogenases to the water-insoluble blue compound formazan, depending on the viability of cells.

HeLa cells (4 ×104 in each well) were incubated in 96-well plates for 24 hours in the presence or absence of essential oil. Twenty microlitres MTT solution (Sigma) (5 mg mL-1 in PBS) were added to each well. The plate was incubated for 4 h at 37°C in a CO2-incubator. One hundred and eighty microlitres of medium was removed from every well without disturbing the cell clusters. A 180 μl methanol/DMSO solution (50:50) was added to each well, and the preparations were mixed thoroughly on a plate shaker with the cell containing formazan crystals. After all of the crystals were dissolved, the A570 values were determined with a microplate reader (ELx 800).

Results and Discussion

Antimicrobial assays

The antimicrobial activities of M. vulgare essential oil against microorganisms examined in the present study and their potency were qualitatively and quantitatively assessed by the presence or absence of inhibition zones and zone diameter (DD), the medium inhibitory concentration (IC50) and the minimal inhibitory concentration (MIC) values. This essential oil displayed varied antibacterial and antifungal activities across the studied pathogens. As can be seen from Table 2, essential oil inhibited the growth of bacterial strains produced a zone diameter of inhibition from 6.6 to 25.2 mm for Gram (+) bacteria, along with IC50 and MIC values ranging from 560-1100 μg/ml and 1120-2600 μg/ml, respectively. Whereas, for Gram (-) bacteria, no antimicrobial activities of essential oil tested against all strains (Pseudomonas aeruginosa 27853, Klebsielle pneumoniae WHO24, Escherichia coli 25922 and Salmonella) has been revealed. Among Gram (+) bacteria, the strongest activity of M. vulgare essential oil was observed against Staphylococcus epidermidis (25.2 mm) followed by Staphylococcus aureus 25923 (18 mm), Enterobacter cloacae (13.8 mm), Bacillus subtilis (13.2 mm), Micrococcus luteus (12 mm) and Staphylococcus aureus 1327 (12 mm). However, Enterococcus faecalis (9.6 mm) and Bacillus cereus (6.6 mm) exhibited moderate to weak activities, respectively.
Table 2

Antibacterial and antifungal activity of the essential oil of M. vulgare using agar disc diffusion, IC50 and minimal inhibition concentration (MIC).

Strains

DDa

IC50b

MICc

DDd

Bacterial strains Gram (+)

    

Staphylococcus aureus

12.0 ± 0.5

2500 ± 50

>1100.00

20 ± 0.5

Staphylococcus epidermidis

25.2 ± 0.3

2200 ± 50

>590.00

26 ± 0.5

Micrococcus luteus

12.0 ± 0.5

1120 ± 50

>560.00

20 ± 1.5

Enterococcus faecalis

9.6 ± 1.0

1140 ± 10

>680.00

25 ± 1.0

Enterobacter cloacae

13.8 ± 0.6

1500 ± 90

>670.00

21 ± 1.4

Staphylococcus aureus 25923

18 ± 1.0

2600 ± 80

>1150.00

24 ± 0.5

Bacillus subtilis

13.2 ± 1.0

2130 ± 50

>890.00

26 ± 0.6

Bacillus cereus

6.6 ± 0.4

2113 ± 40

>950.00

21 ± 1.0

Bacterial strains Gram (-)

    

Botrytis cinerea

NS

NS

NS

21 ± 0.5

Pseudomonas aeruginosa 27853

NS

NS

NS

20 ± 1.0

Klebsielle pneumoniae WHO24

NS

NS

NS

21 ± 0.9

Escherchia coli 25922

NS

NS

NS

22 ± 0.8

Salmonella

NS

NS

NS

29 ± 1.0

Fungal strains

    

Botrytis cinerea

12.6 ± 0.5

2190 ± 12

>1100.00

29 ± 1.0

Fusarium solani

6.9 ± 0.5

3220 ± 20

>1190.00

28 ± 0.6

Penicillium digitatum

6.6 ± 0.4

3200 ± 15

>1120.00

21 ± 0.9

Aspergillus. niger

6.4 ± 0.0

3000 ± 50

>1180.00

30 ± 0.5

Results are means of three different experiments,

a DD: Disc Diameter of inhibition (halo size) in (mm), E. oil 100 μg/disc,

b MIC: minimum inhibitory concentration (μg/ml),

cIC50: 50% inhibition concentration (μg/ml),

dDD: Disc Diameter of inhibition zone of ampicillin (10 μg/disc) and cycloheximide (10 μg/disc), were used as positive controls for bacteria and fungi, respectively,

NS: not sensitive.

For the fungi strains, the disc diameter zones of inhibition ranged from 6.4-12.6 mm, along with IC50 and MIC values ranging from 2190-3000 μg/ml and 1100-1180 μg/ml, respectively. The maximal inhibition zones was obtained for Botrytis cinerea, however Fusarium solani, Penicillium digitatum and Aspergillus niger exhibited weak activity. Our results suggest that Gram (+) bacteria are more sensitive to the M. vulgare essential than Gram (-) bacteria. This was consistent with the previous studies on other spices and herbs [38, 39]. This generally higher resistance among Gram (-) bacteria could be ascribed to the presence of their outer membrane, surrounding the cell wall, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. The absence of this barrier in Gram (+) bacteria allows the direct contact of the essential oil's hydrophobic constituents with the phospholipids bilayer of the cell membrane, causing either an increase of ion permeability and leakage of vital intracellular constituents, or impairment of the bacterial enzyme systems. Differences in MIC values of bacteria may be related to the differential susceptibility of bacterial cell wall, which is the functional barrier to minor differences present in the outer membrane in the cell wall composition [40]. The highest sensitivity of Staphylococcus epidermidis and Staphylococcus aureus may be due to its cell wall structure and outer membrane.

It is most likely that numerous components of the essential oils play a crucial role in defining the features of oils, lipophilic or hydrophilic attraction and fixation on cell wall and membranes, and cellular distribution [41]. This feature is very important because, depending on their component, the distribution of the oil in the cell determines the different types of biological activities such as antibacterial, antifungal and cytotoxicity. As reported previously [10], the M. vulgare essential oils isolated by hydrodistillation from the aerial parts was analyzed by HP-5MS column. The general chemical composition of the essential oil, the percentage contents, and retention indices of the components are given in Table 3. Thirty four components could be identified in the oil (100% of the total oil). The essential oil is constituted mainly by approximately equal amounts of oxygenated monoterpenes (40.02%) and sesquiterpenes hydrocarbons (42.70%). The major components were γ-eudesmol (11,93%), followed by β- citronellol (9,90%), Citronellyl formate (9,50%) and germacrene D (9,37%). When compared with previous studies [1621], our study showed that this essential oil possessed an original composition with the main component of γ-eudesmol (11.93%), which is not observed elsewhere.
Table 3

Chemical composition, retention indices and percentage composition of the M. vulgare essential oil.

Compound

RI

%

Identification

1

N-trimethylsilyl trifluoroacetamide

764

2.35

MS, RI

2

N, N-bis trimethylsilyl trifluoroacetamide

857

0.97

MS, RI

3

α- pinene

932

1.16

MS, RI

4

Camphene

948

0.49

MS, RI

5

1,8-Cineole

1044

3.72

MS, RI

6

α-thujone

1131

2.29

MS, RI

7

1-Vinylcyclohexane

1143

0.75

MS, RI

8

Camphor

1174

1,03

MS, RI

9

Iso menthone

1197

0.57

MS, RI

10

Borneol

1199

0.61

MS, RI

11

β-citronellol

1266

9.90

MS, RI

12

Geraniol

1295

2.74

MS, RI

13

Citronellyl formate

1315

9.50

MS, RI

14

Geranyl formate

1344

6.25

MS, RI

15

α-copaene

1419

1.37

MS, RI

16

β-Bourbonene

1429

1.96

MS, RI

17

trans-caryophyllene

1462

2.15

MS, RI

18

α-Muurolene

1484

0.63

MS, RI

19

α-amorphene

1490

0.81

MS, RI

20

α-Humulene

1495

0.68

MS, RI

21

Neoalloocimene

1502

0.91

MS, RI

22

neryl acetate

1512

3.41

MS, RI

23

Germacrene-D

1521

9.37

MS, RI

24

Ledene

1534

5.35

MS, RI

25

β-bisabolene

1544

0.86

MS, RI

26

δ-cadinene

1559

3.30

MS, RI

27

α -agarofuran

1581

0.42

MS, RI

28

Furan-2-one, 4-phenyltetrahydro

1616

1.44

MS, RI

29

γ-Eudesmol

1647

11.93

MS, RI

30

β-Cubebene

1674

1.52

MS, RI

31

Citronellyl butanoate

1682

0.66

MS, RI

32

Geranyl tiglate

1712

5.53

MS, RI

33

Cyclononasiloxane, octadecamethyl

2198

3.08

MS, RI

34

Eicosamethylcyclodecasiloxane

2264

2.29

MS, RI

Total identification

 

100

 

Yield (g/100 g dry weight)

 

00.34

 

Hydrocarbon monoterpenes

 

01.65

 

Oxygenated monoterpenes

 

40.02

 

Hydrocarbon sesquiterpenes

 

42.70

 

Oxygenated sesquiterpene

 

06.19

 

The antimicrobial properties of essential oils from aerial part of M. vulgare are suspected to be associated, in part with their high contents of oxygenated compounds (46.21%). Several researchers also report mono- and sesquiterpenoids as the major components of essential oils which are phenolic in nature [42, 43]. It is therefore reasonable to assume that their antimicrobial activity might be related to the abundance of phenolic compounds. Referring to the literature, the antimicrobial activity of the tested essential oil can be related to the contribution of the mixture between major (γ-eudesmol, β- citronellol, citronellyl formate and germacrene D) and minor (camphene, borneol) [10] constituents, which known to have efficient antimicrobial properties [44, 45]. In addition, germacrene-D is known to have a strong effect on insect behavior [46] and has significant antibacterial and antifungal activities [47]. Therefore, Essential oils always represent a complex mixture of different chemical components, thus it is very difficult to reduce the antibacterial effect of the total oil to a few active principles.

Compared to the positive control, ampicillin (belonging to the penicillin group of beta-lactam antibiotics is able to penetrate Gram positive and some Gram negative bacteria) and cycloheximide (generally used only in in vitro research applications as a fungicide, and is not suitable for human use as a therapeutic compound) were found to possess lower activity than the tested essential oil. We explain this by the fact, that pure component, such as antibiotics give a more potent antimicrobial activity when compared to a complex mixture of components such as essential oils.

Cytotoxicity assays

The effect of different concentrations (3.91-3000 μg/ml), of M. vulgare essential oil on HeLa cell lines were studied. As depicted in Table 4, they significantly decreased the viability of HeLa in a dose dependent manner. For a concentration up to 250 μg/ml, essential oil destructed HeLa cells by 27%, however for a concentration higher than 500 μg/ml, all HeLa cells were destructed. At lower doses, the oil was tolerated by the cells and its IC50 (the concentration of the tested oil required to reduce the cell survival fraction to 50% of the control) was 0.258 μg/ml. The volatile oil displayed an excellent cytotoxic effect towards the human tumor cell line. This makes the tested oil certainly deserve some further investigation. Referring to the literature, the cytotoxicity of the tested essential oils may be due to the presence of some monoterpenes and sesquiterpenes including α-Humulene [48] and isoprenoids including geraniol [49]. These compounds were reported to be active against the tumor cell lines, but in our oil, they exist in small amount [10]. Although, the presence of germacrene D in a good amount can enhances the cytoxicity of the tested oil. This results was confirmed by Setzer et al., who reported that germacrene D has exhibited a 7-fold stronger cytotoxic activity against the Hs 578T cell line in comparison with α-pinene and limonene and a 6-fold stronger action against Hs 578T and Hep-G2 than 1,8-cineole, linalool, 4-terpineol and α-terpineol [50]. Some reports support the relationship of cytotoxicity with antioxidant activity [51]. So the antioxidant activity of M. vulgare essential oil might contribute to its cytotoxic activity [45]. Finally, we noted that the synergistic effects of these active chemicals with other constituents of the essential oil should be taken into consideration. This study also justifies and reinforces the use of this plant on traditional medicine. Although all in vitro experiments hold limitations with regards to possible in vivo efficacy. The results of this study are very promising as it will serve as a data base for researchers in these kinds of studies for indicating which essential oils and plant oils may be useful for specific medical conditions. Furthermore, it is still necessary to investigate in vivo bioactivity and cytoxicity of the oil, to explore in more depth its potential beneficial use in diseases and infections caused by microbes.
Table 4

Cytotoxic activity of M. vulgare essential oil determined by the MTT assay.

Oil (μg/ml)

Mean OD570 (nm)

% Viable HeLa cell line

0

0.971

100.00

3.91

0.836

86.10

7.81

0.815

83.93

15.63

0.798

82.18

31.25

0.780

80.94

62.50

0.756

77.85

125

0.717

73.84

250

0.707

72.81

500

0.015

01.54

1000

0.031

00.31

3000

0.023

00.21

Conclusion

In conclusion, our study can be considered as the first report on the antimicrobial and cytotoxic properties of M. vulgare volatile oil. The in vitro antimicrobial activity of the obtained may well be due to the presence of synergy, antagonism or additive effects of the tested major components of the oils, which possess various potency of activity. The results of the cytotoxic activity against HeLa cell lines in this study are very promising with regards to possible antineoplastic chemotherapy and form a very sound basis for future research. Our results are a contribution to a better valorization of this medicinal plant. Several other biological tests will be worthwhile to search for more eventual activities of this plant to characterize active principles, and assess toxicity by laboratory assays.

Declarations

Authors’ Affiliations

(1)
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, BPW, University of Sfax
(2)
Laboratoire de Chimie des Substances Naturelles, Faculté des Sciences de Sfax, University of Sfax
(3)
Laboratoire de Microbiologie Alimentaire, Faculté des sciences de Sfax, University of Sfax
(4)
Unité de recherche Biotechnologie et pathologies, Institut Supérieur de Biotechnologie de Sfax, University of Sfax
(5)
Département de Génie des procédés, ISET Sfax, University of Sfax
(6)
Laboratoire de Biochimie, Faculté des sciences de Sfax, University of Sfax

References

  1. Wink M: Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry. 2003, 64: 3-19. 10.1016/S0031-9422(03)00300-5View ArticlePubMedGoogle Scholar
  2. Chopra RN, Nayer SL, Chopra IC: Glossary of Indian Medicinal Plants, CSIR, V ed. New Delhi. 1956, 12: 157-Google Scholar
  3. Roman RR, Aharcon AF, Lara LA, Flores SJL: Hypoglycemic effect of plants used in Mexico as antidiabetics. Arch Med Res. 1992, 23 (1): 59-64.Google Scholar
  4. El-Bardai S, Morel N, Wibo M, Fabre N, Llabres G, Lyoussi B, Quetin L: The vasorelaxant activity of marrubenol and marrubiin from Marrubium vulgare. Plant Med. 2003, 69 (1): 75-77. 10.1055/s-2003-37042.View ArticleGoogle Scholar
  5. El-Bardai S, Lyoussi B, Wibo M, Morel N: Comparative Study of the antihypertensive activity of Marrubium Vulgare and of the dihydropyridine calcium antagonist amlodipine in spontaneously hypertensive rat. Clin Exp Hyprtens. 2004, 26 (6): 465-474. 10.1081/CEH-200031818.View ArticleGoogle Scholar
  6. DeSouza MM, DeJesus RAP, Cechinel-Filho V, Schlemper V: Analgesic profile of hydroalcoholic extract obtained from Marrubium vulgare. Phytomed. 1998, 5 (2): 103-107.View ArticleGoogle Scholar
  7. Sahpaz S, Garbacki N, Tits M, Bailleul F: Isolation and pharmacological activity of phenylpropanoid esters from Marrubium vulgare. J Ethnopharmacol. 2002, 79 (3): 389-392. 10.1016/S0378-8741(01)00415-9View ArticlePubMedGoogle Scholar
  8. Schlemper V, Ribas A, Nicolau M, Cechinel-Filho V: Antispasmodic effects of hydroalcoolic extract of Marrubium vulgare on isolated tissues. Phytomedicine. 1996, 211-216. 3.Google Scholar
  9. Weel KCG, Venskutonis PR, Pukalskas A, Gruzdiene D, Linssen JPH: Antioxidant activity of horehound (Marrubium vulgare) grown in Lithuania. Fett/Lipid. 1999, 101 (10): 395-400. 10.1002/(SICI)1521-4133(199910)101:10<395::AID-LIPI395>3.0.CO;2-L.View ArticleGoogle Scholar
  10. Kadri A, Zarai Z, Bekir A, Gharsallah N, Damak M, Gdoura R: Chemical composition and antioxidant activity of Marrubium vulgare L. essential oil from Tunisia. African Journal of biotechnolgy. 2011, 10 (198): 3908-3914.Google Scholar
  11. Stulzer HK, Tagliari MP, Zampirolo JA, Cechinel-Filho V, Schlemper V: Antioedematogenic effect of marrubiin obtained from Marrubium vulgare. J Ethnopharmacol. 2006, 108 (3): 379-392. 10.1016/j.jep.2006.05.023View ArticlePubMedGoogle Scholar
  12. Baratta MT, Dorman HJD, Deans SG, Figueiredo AC, Barroso JG, Ruberto G: Antimicrobial and antioxidant properties of some commercial essential oils. Flav Fragr J. 1998, 13: 235-44. 10.1002/(SICI)1099-1026(1998070)13:4<235::AID-FFJ733>3.0.CO;2-T.View ArticleGoogle Scholar
  13. Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V, Arzedi E Palmas F: In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Letters in Applied Microbiology. 1999, 29: 130-135. 10.1046/j.1472-765X.1999.00605.xView ArticlePubMedGoogle Scholar
  14. Bounatirou S, Smiti S, Miguel MG, Faleiro L, Rejeb MN, Neffati M, Costa MM, Figueiredo AC, Barroso JG, Pedro LG: Chemical composition, antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. Et Link. Food Chem. 2007, 105: 146-155. 10.1016/j.foodchem.2007.03.059.View ArticleGoogle Scholar
  15. Fabio A, Cermelli C, Fabio G, Nicoletti P, Quaglio P: Screening of the antibacterial effects of variety of essential oils on microorganisms responsible for respiratory infections. Phytother Res. 2007, 21: 374-377. 10.1002/ptr.1968View ArticlePubMedGoogle Scholar
  16. Weel KCG, Venskutonis PR, Pukalskas A, Gruzdiene D, Linssen JPH: Antioxidant activity of horehound (Marrubium vulgare) grown in Lithuania. Fett/Lipid. 1999, 101 (10): 395-400. 10.1002/(SICI)1521-4133(199910)101:10<395::AID-LIPI395>3.0.CO;2-L.View ArticleGoogle Scholar
  17. Nagy M, Svajdlenka E: Comparison of Essential Oils from Marrubium vulgare L. and M. peregrinum L. J Essent Oil Res. 1998, 10: 585-587.View ArticleGoogle Scholar
  18. Saleh MM, Glombitza KW: Volatile Oil of Marrubium vulgare and its Anti-schistosomal Activity. Planta Med. 1989, 55: 105-108.View ArticleGoogle Scholar
  19. Morteza-Semnani K, Saeedi M: The Essential Oil Composition of Marrubium astracanicum Jacq. From Iran. J Essent Oil Bearing Plants. 2004, 7: 239-242.View ArticleGoogle Scholar
  20. Asadipour A, Mehrabani M, Nazeri V, Tabarraii M: Composition of the essential oil of Marrubium vulgare L. Ulum-i-Daroei. 2005, 2: 77-82.Google Scholar
  21. Khanavi M, Ghasemian L, Hosseiny Motlagh E, Hadjiakhoondi A, Shafiee A: Chemical Composition of The Essential Oils of Marrubium parviflorum Fisch. & C.A. Mey and Marrubium vulgare L. from Iran. Flav Fragr J. 2005, 20: 324-326. 10.1002/ffj.1425.View ArticleGoogle Scholar
  22. Voravuthikunchai SP, Sririrak T, Limsuwan S, Supawita T, Iida T, Honda T: Inhibitory effects of active compounds from Punica granatum pericarp on Verocytotoxin by enetrohaemorrhagic Escherichia coli 0157: H7. J Health Sci. 2005, 51 (5): 590-596. 10.1248/jhs.51.590.View ArticleGoogle Scholar
  23. Burt S: Essential oils: their antibacterial properties and potential applications in foods. International Journal of Food Microbiology. 2004, 94: 223-253. 10.1016/j.ijfoodmicro.2004.03.022View ArticlePubMedGoogle Scholar
  24. Berghe VA, Vlietinck AJ: Screening methods for antibacterial and antiviral agents from higher plants. Method for Plant Biochemistry. 1991, 6: 47-68.Google Scholar
  25. Wade D, Silveira A, Rollins-Smith L, Bergman T, Silberring J, Lankinen H: Hematological and antifungal properties of temporin A and a cecropin A-temporin A hybrid. Acta Biochim Pol. 2001, 48: 1185-1189.PubMedGoogle Scholar
  26. Pérez C, Agnese AM, Cabrera JL: The essential oil of Senecio graveolens (Compositae): chemical composition and antimicrobial activity tests. J Ethnopharmacol. 1999, 66: 91-96. 10.1016/S0378-8741(98)00204-9View ArticlePubMedGoogle Scholar
  27. Bagamboula CF, Uyttendaele M Debevere J: Acid tolerance of Shigella sonnei and Shigella flexneri. J Appl Microbiol. 2002, 93 (3): 479-486. 10.1046/j.1365-2672.2002.01714.xView ArticlePubMedGoogle Scholar
  28. Saïdana D, Mahjoub S, Boussaada O, Chriaa J, Mahjoub MA, Chéraif I, Daami M, Mighri Z, Helal AN: Antibacterial and Antifungal Activities of the Essential Oils of two Saltcedar Species from Tunisia. J Am Oil Chem Soc. 2008, 85 (9): 817-826. 10.1007/s11746-008-1251-9.View ArticleGoogle Scholar
  29. Hichri F, Ben Jannet H, Cheriaa J, Jegham S, Mighri Z: Antibacterial activities of a few prepared derivativesof oleanolic acid and of other natural triterpenic compounds. CR Chim. 2003, 6: 473-483. 10.1016/S1631-0748(03)00066-3.View ArticleGoogle Scholar
  30. Bel Haj Khether F, Ammar S, Saidana D, Daami-Remadi M, Cheriaa J, Liouane K, Mahjoub MA, Helal AN, Mighri Z: Chemical composition, antibacterial and antifungal activities of Trichoderma sp. growing in Tunisia. Annals of Microbiology. 2008, 58: 303-308. 10.1007/BF03175334.View ArticleGoogle Scholar
  31. May J, Chan CH, King A, Williams L, French GL: Time-kill studies of tea tree oils on clinical solates. J Antimicrobial Chemoter. 2000, 45: 639-643. 10.1093/jac/45.5.639.View ArticleGoogle Scholar
  32. Ferreira A, Proenca AC, Serralheiro CMLM, Araujo MEM: The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J Ethnopharmacology. 2006, 108: 31-37. 10.1016/j.jep.2006.04.010.View ArticlePubMedGoogle Scholar
  33. Saidana D, Mahjoub MA, Boussaada O, Chriaa J, Cheraif I, Daami-Remadi M, Mighri Z, Helal AN: Chemical composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae). Microbiology Res. 2008, 163: 445-455. 10.1016/j.micres.2006.07.009.View ArticleGoogle Scholar
  34. Boussaada O, Ammar S, Saidana D, Chriaa J, Chraif I, Daami-Remadi M, Helal AN, Mighri Z: Chemical composition and antimicrobial activity of volatile components from capitula and aerial parts of Rhaponticum acaule DC growing wild in Tunisia. Microbiology Res. 2008, 163: 87-95. 10.1016/j.micres.2007.02.010.View ArticleGoogle Scholar
  35. Hammer KA, Carson CF, Riley TV: Antimicrobial activity of essential oils and other plant extracts. J App Microbiology. 1999, 86: 985-990. 10.1046/j.1365-2672.1999.00780.x.View ArticleGoogle Scholar
  36. Delaquis PJ, Stanich K, Girard B, Mazza G: Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int J Food Microbiology. 2002, 74: 101-109. 10.1016/S0168-1605(01)00734-6.View ArticleGoogle Scholar
  37. Omar-Hamza JM, Carolien JP, Van Den Bout-van Den Beukel, MeckyMatee IN, Paul-Verweij JAME: Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections. J Ethnopharmacology. 2006, 108: 124-132. 10.1016/j.jep.2006.04.026.View ArticleGoogle Scholar
  38. Lopez P, Sanchez C, Batlle R, Nerin C: Solid- and vapor-phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains. Journal of Agricultural and Food Chemistry. 2005, 53: 6939-6946. 10.1021/jf050709vView ArticlePubMedGoogle Scholar
  39. Ceylan E, Fung DYC: Antimicrobial activity of spices. Journal of Rapid Methods and Automation in Microbiology. 2004, 12: 1-55. 10.1111/j.1745-4581.2004.tb00046.x.View ArticleGoogle Scholar
  40. Zhao WH, Hu ZO, Okubo S, Hara Y, Shimamura T: Mechanism of synergy between epigallocatechin gallate and β-lactams against methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 2001, 45: 1737-1742. 10.1128/AAC.45.6.1737-1742.2001.View ArticleGoogle Scholar
  41. Cal K: Skin penetration of terpenes from essential oils and topical vehicles. Planta Med. 2006, 72: 311-316. 10.1055/s-2005-916230View ArticlePubMedGoogle Scholar
  42. Oyedeji O, Afolayan A: Comparative study of the essential oil composition and antimicrobial activity of Leonotis leonurus and L. ocymifolia in the Eastern Cape. African J Bot. 2005, 71: 114-116.View ArticleGoogle Scholar
  43. Cakir A, Kordali S, Zengin H, Izumi S, Hirata : Composition and antifungal activity of essential oils isolated from Hypericum hyssopifolium and Hypericum heterophyllum. Flavour Frag J. 2004, 19: 62-68. 10.1002/ffj.1279.View ArticleGoogle Scholar
  44. Nada B, Mirjana S, Valerija D: Phytochemical composition and antimicrobial activity of Satureja montana L. and Satureja cuneifolia Ten. essential oils. Acta Bot Croat. 2005, 64: 313-322.Google Scholar
  45. Cimanga K, Kambu K, Tona L, Apers S, De Bruyne T, Hermans N, Totte J, Pieters L, Vlietinck AJ: Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J Ethnopharmaco. 2002, 79: 213-220. 10.1016/S0378-8741(01)00384-1.View ArticleGoogle Scholar
  46. Picaud S, Olsson ME, Brodelius M, Brodelius PE: Cloning, expression, purification and characterization of recombinant (+)- germacrene D synthase from Zingiber officinale. Arch Biochem Biophys. 2006, 452: 17-28. 10.1016/j.abb.2006.06.007View ArticlePubMedGoogle Scholar
  47. Sahin F, Gulluc M, Daferera D, Sokmen A, Sokmen M, Polissiou M, Agar G, Ozer H: Biological activities of the essential oils and methanol extract of Origanum vulgare ssp. vulgare in the eastern Anatolia region of Turkey. Food Control. 2004, 15: 549-557. 10.1016/j.foodcont.2003.08.009.View ArticleGoogle Scholar
  48. Sylvestre M, Legault J, Dufour D, Pichette A: Chemical composition and anticancer activity of leaf essential oil of Myrica gale L. Phytomedicine. 2005, 12: 299-304. 10.1016/j.phymed.2003.12.004View ArticlePubMedGoogle Scholar
  49. Burke YD, Stark MJ, Roach SL, Sen SE, Crowell PL: Inhibition of pancreatic cancer growth by the dietary isoprenoids farnesol and geraniol. Lipids. 1997, 32: 151-156. 10.1007/s11745-997-0019-yView ArticlePubMedGoogle Scholar
  50. Setzer WN, Schmidt JM, Noletto JA, Vogler B: Leaf oil compositions and bioactivities of abaco bush medicines. Pharmacology online. 2006, 3: 794-802.Google Scholar
  51. Hou J, Sun T, Hu J, Chen SY, Cai XQ, Zou GL: Chemical composition, cytotoxic and antioxidant activity of the leaf essential oil of Photinia serrulata. Food Chem. 2007, 103: 355-358. 10.1016/j.foodchem.2006.07.060.View ArticleGoogle Scholar

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