Volume 02, Issue 12 (December 2013)

Chemical Composition and Antibacterial Activity of Essential Oils of Two Aromatic Plants : Mentha Spicata and Lippia Citriodora Irrigated by Urban Wastewater

DOI : 10.17577/IJERTV2IS120611

Download Full-Text PDF Cite this Publication
Fatima Bensabah, Houda Sbayou, Souad Amghar, Abdeslam Lamiri, Jamal Naja, 2013, Chemical Composition and Antibacterial Activity of Essential Oils of Two Aromatic Plants : Mentha Spicata and Lippia Citriodora Irrigated by Urban Wastewater, INTERNATIONAL JOURNAL OF ENGINEERING RESEARCH & TECHNOLOGY (IJERT) Volume 02, Issue 12 (December 2013),

Text Only Version

Chemical Composition and Antibacterial Activity of Essential Oils of Two Aromatic Plants : Mentha Spicata and Lippia Citriodora Irrigated by Urban Wastewater

Fatima Bensabap, Houda Sbayou2, Souad Amghar2, Abdeslam Lamiri1 And Jamal Naja1

1: Laboratory of Applied Chemistry and Environment, Faculty of Science and Technologies, University Hassan 1, Km 3, B.P. 577, Settat, Morocco.

2: Laboratory of agrofood and health, Faculty of sciences and technologies, University Hassan 1, Settat Km 3, B.P: 577, Settat, Morocco.

Abstract

The objective of this study is the valorization of essential oils (EO) of two aromatic and medicinal plants: Mentha spicata and Lippia citriodora irrigated by urban wastewater from the city of Settat-Morocco. valorization consisite to test their antibacterial activity. Their chemical composition was studied by chromatography-mass spectrometry gas (GC/MS). These EOs were tested against three bacterial strains: Escherichia coli ATCC 25922, Streptococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 ATCC. Our study showed that these essential oils from Menthe Spicata have significant antibacterial activity against the three species of bacteria studied (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853). Also, it is found that fresh leaves of Mentha spicata was the most active of all tested bacteria and the activity of this plant remains effective even when

irrigated with wastewater, this is due to the presence of carvone (57,11%) and limonene (27,77%). On the other side, essential oils from Lippia citriodora, have not inhibitory activity against Pseudomonas aeruginosa, and fresh leaves of Lippia Citriodora have a more inhibitory activity when, its irrigated with wastewater.Activity of fresh leaves of Lippia Citriodora irrigated by wastewater is due to the presence of neral (14,34%), geranial (14,75%) and limonene (25,86%).Generally, the irrigation by urban wastewater did not affect the MBC/MIC ratio, since this ratio values are the same for all of the essential oils value, indicating a bactericidal effect of the 5 oils tested under different conditions.

Key words : Mentha spicata, Lippia citriodora, essential oil, antibacterial activity, urbain wastewater.

  1. Introduction

    Medicinal plants have been traditionally used for pharmaceutical and dietary therapy in long history [1]. and pharmaceutical industries [2]. Essential oils are generally used in the cosmetic, medical and food industries. Antibiotic resistance has become a global concern [3].Aromatic and medicinal plants had acquired particular attention in the field of intensive research on the natural antimicrobial com-pounds. They constitute a constant source of active reagents against pathogen germs [4]. Among these products, essential oils (EOs) produced by aromatic plants as secondary metabolites, have gained a net interest by many investigators [5, 6, 7 and 8]

    EOs are volatile natural complex compounds characterized by strong odour [9], and represent very complex natural mixtures which may contain more than sixty individual components at quite different concentrations [10]. Major components can constitute up to 85% of the EOs, whereas other components are present only as trace [6]. It has been recognized that some EOs have antimicrobial, antifungal, anticancer and antioxidants properties [11, 12, 13, 14, 15 and 16]

    Mentha spicata also known as spearmint belongs to the Lamiaceae (labiatae) family. The genus Mentha consists 2530 species; and spearmint is the most common among them [17]. The plant is a glabrous perennial with creeping rhizomes [18]. Mint oil is of economic importance and is widely used in pharmaceutical, cosmetic, food, confectionary and beverage industries [19]. Spearmint oil contains monoterpenoids like carvone, limonene, dihydrocarveol and s-carvone. Some of them were found to possess high antioxidant activity than -tocopherol [20]. The plant is also known for its ability to enhance memory [21]. Apart from being a stimulant and carminative, the mint plant is also known for its insecticidal, antimicrobial, antispasmodic and antiplatelet properties [22, 23, 24 and 25].

    The genus Lippia (Verbenaceae) includes approximately 200 species of herbs, shrubs and small trees [26]. Most of them are traditionally utilized as remedies for gastrointestinal and respiratory problems.Some species have shown antimalarial, antiviral and cytostatic properties. It is believed that their essential oils and phenolic compounds (flavonoids) are responsible for these properties [27]. It is cultivated mainly due to the lemon-like aroma

    emitted from its leaves that are utilized for the preparation of herbal tea, which is reputed to have antispasmodic, antipyretic, sedative and digestive properties [28, 27, 29 and 30]. Lemon verbena has a long history of folk uses in treating asthma, spasms, cold, fever, flatulence, colic, diarrhea, indigestion, insomnia and anxiety [28 and 29]. The essential oil from its leaves has been shown to exhibit antimicrobial activity [31, 32, 33, 34 and 35]..

  2. Materials and methods

    1. Plant material

      Plantation was in May 2009 at experimental plots located in the Faculty of Science and Technics of Settat- Morocco (FSTS). The irrigations were made by urban wastewater purified by lagoon from Settat city (Morocco) and water well located in FSTS considered as a witness. Harvests for fresh leaves were made by hand. Water used for irrigation were characterized physico-chemical analyzes are determined previous work [36].

    2. Essential oil extraction

      The chemical composition of essential oils was also determined using fresh and dried leaves of Lippia Citriodora and Mentha spicata harvested.

      Distillation apparatus consisted of a heating cap, a 1.5 L extraction flask, a cooling system and a receiver for hydro distillate. Thirty grams of plant leaves and 800 mL of water were used and the distillation was carried out for 3 h after the mixture reached boiling at 100 °C. The essential oil obtained was dried under anhydrous sodium sulfate and stored at 4 °C in the dark. Each oil, after extraction has been divided into two parts, one was used for chemical analysis, while the second was used to study the antibacterial tests.

      The following table presents the five essential oils used.

      Table1: The essential oils used in this study.

      Essential oil

      Aromatic plant

      Type leaves

      Irrigation water

      Mentha (1)

      Mentha spicata

      fresh leaves

      well water

      Mentha (2)

      Mentha spicata

      fresh leaves

      wastewater

      Lippia (1)

      Lippia Citriodora

      fresh leaves

      well water

      Lippia (2)

      Lippia Citriodora

      fresh leaves

      wastewater

      Lippia (3)

      Lippia Citriodora

      dried leaves

      Wastewater

    3. Gas chromatography-mass spectrometry analysis (GC/MS)

      The essential oil was characterized using a gas chromatograph Trace GC Ultra equipped with an autoinjector (Triplus) directly interfaced with a mass spectrophotometer with a flame ionization detector (Pdains Q). Capillary column was DB-5 (5% of diphenyl and 95% of dimethypolysiloxane), 30m in length, 0.25mm thickness. Separation conditions were : 50°C for 2mn, 50200°C at 5°C/min. Temperature of the injector was 220°C. The volume injected was 0.1

      µL. The carrier gas was helium with a flow rate of 1.4 ml.min1. The oil constituents were identified by comparison of their retention indices and their mass spectra with those of authentic samples. Quantitative analysis (in percent) was performed by peak area measurement.

    4. Bacterial strains

      The essential oils were tested against the following bacteria: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. All strains were obtained from Prof. Angeles Manresa Microbiology laboratory, Faculty of pharmacy, University of Barcelona. Spain.

    5. Disc diffusion method

      The agar disc diffusion method was employed for the determination of antibacterial activities of the tested EO as described previously [7] with some modifications.

      The test was performed in sterile Petri plates containing Muller Hinton Agar. Sterile paper disk (6 mm in diameter) were impregnated with 10 µL of EO and were placed on the Petri plates previously inoculated with a microbial suspension adjusted to 106 CFU/ mL. Standard antibiotics amoxicillin (25 µg) were used as positive control. The Petri plates were incubated at 37°

      C for 24h. The diameters of inhibition zones were measured in millimeters. Tests were carried out in triplicate.

    6. Determination of MIC and MBC

      MIC (Minimal Inhibition Concentration) was determined by the method of micro-broth dilution [37]. A serial of dilution of essential oil ranging from 10

      µL/mL to 0.15 µL/mL were prepared in test tubes containing Muller Hinton medium with 0.15% Agar [38]. Each tube was inoculated with a bacterial suspension adjusted to 106 CFU/mL. The MIC was the lowest concentration for which no growth was detected after 24h at 37° C [39]. While for the determination of MBC (Minimum Bactericidal Concentration), 10 µL of the tubes showing no growth were subcultured on nutrient agar plates and the Petri plates were incubated for 24h at 37° C. The MBC was the lowest concentration of the EO at which no growth occurred on the plates [40]. Each assay was repeated thrice

  3. RESULTS AND DISCUSSION

    1. Chemical composition of the essential oils

      1. Essential oils of Mentha spicata

        The Table 2 shows the major compounds of the two essential oils of Mentha spicata

        Table 2. Chemical composition (%) of two essential oils of Mentha spicata.

        Compounds

        Retention time

        Mentha (1)

        Mentha (2)

        -Pinene

        8.59

        0,76

        0,69

        Camphene

        9.06

        0,20

        Trace

        3-Carene

        10.04

        1,33

        1,01

        -Phellandrene

        10.67

        0,81

        0,60

        Limonene

        12.01

        31,13

        27,77

        1,8-Cineole

        14.59

        0,25

        0,28

        Cis-Ocimene

        15.88

        0,29

        Trace

        Borneol

        16.85

        0,56

        0,84

        Sabinene hydrate

        17.28

        Trace

        0,39

        Isocyclocitral

        17.94

        0,46

        1,71

        Trans-Carveol

        18.87

        3,90

        1,79

        Pulegone

        19.42

        0,33

        0,30

        Carvone

        19.74

        53,69

        57,11

        Bornyl acetate

        21.63

        0,26

        0,27

        iso-Limonene

        22.79

        0,22

        trace

        -Bourbonene

        24.32

        0,68

        0,71

        Caryophyllene

        25.12

        0,40

        Absent

        Aromandrene

        25.40

        0,67

        0,50

        Germacrene D

        27.31

        0,87

        0,65

        Calamenene

        27.78

        0,22

        0,22

        Mentha spicata irrigated by well water contained carvone (53,69%), and limonene (31,13%) as the major compounds. The oils from Mentha spicata irrigated by wastewater are also characterized by a high percentage of carvone (57,11%), andlimonene (27,77%).

        The chemical composition of two EOs of the Mentha spicata is qualitatively similar, although there are some differences in the concentrations of the compounds.

        The biosynthesis of limonene and carvone in Mentha spicata are shown in previous work [36].

        The spearmint essential oil was characterized by the dominant presence of carvone (53.69%) from fresh leaves of Mentha spicata irrigated by well water , in agreement with other authors, such as [41], who identified carvone occurring at 68.4% in samples collected in Greece [42], who found it at 76.65% in samples collected in India, and [43], who found it

        at 48.4% in samples collected in Turkey. Also, in essential oil from M. spicata collected in Montene- gro (Europe), [44] reported carvone as the major constituent (49.52%),

      2. Essential oils of Lippia Citriodora

        The Table 3 shows the major compounds of the three essential oils of Lippia Citriodora

        Table 3. Chemical composition (%) of three essential oils of Lippia Citriodora V(1), V(2) and V(3).

        Compounds

        Retention time

        Lippia (1)

        Lippia (2)

        Lippia (3)

        -Pinene

        8.62

        0,94

        –

        1,06

        Sabinene

        10.00

        2,22

        2,33

        2,42

        Limonene

        11.99

        23,39

        25,86

        28,32

        Trans- Ocimene

        12.75

        2,40

        2,67

        –

        Nerol

        17.77

        1,64

        1,50

        1,40

        Neral

        19.55

        15,29

        14,34

        13,85

        Geranial

        20.57

        15,63

        14,75

        14,06

        -Caryophyllene

        25.41

        3,52

        3,98

        3,35

        -Curcumene

        27.37

        9,37

        10,81

        11,36

        Neryl acetate

        27.78

        4,84

        6,64

        2,38

        Copaene

        28.28

        2,60

        3,42

        2,38

        -Cadinene

        28.59

        1,52

        –

        –

        Spathulenol

        30.14

        6,28

        5,43

        8,29

        Caryophyllene oxide

        30.29

        6,37

        4,23

        7,07

        -Cadinene

        31.93

        3,86

        3,75

        3,86

        The main volatile components of fresh leaves lippia Citriodora irrigated by well water are limonene (23,39%), neral (15,29%) and geranial (15,63%), for EOs from fresh leaves of Lippia citriodora irrigated by wastewater , we found limonene (25,86%), neral (14,34%) ad geranial (14,75%) were the main components. And EOs from dried leaves of Lippia

        citriodora irrigated by wastewater, we found limonene (28,32%), neral (13,85%) and geranial (14,06%) were the main components.

        Also the chemical composition of three EOs of the Lippia ciriodora is qualitatively similar, although there are some differences in the concentrations of the

        compounds. These differences are due to irrigation with urban wastewater.

        The chemical composition of the essential oil from the leaves of Lippia Citriodora has also been studied and reviewed [45, 28, 46, 47, 48, 49, 33, 27, 29, 35, 26, 50,

        51 and 52]. The genus Lippia shows a rich genetic diversity, enabling it to synthesize a variety of essential oil constituents in plants grown in different parts of the world [46 and 29]. However, the composition of the essential oil obtained from the same plant stock remains constant under the same environmental conditions [46 and 29].

        For two waters used for irrigation, citral (neral and geranial) and limonene were the dominating compounds in the essential oil of Lippia Citriodora leaves, these results are in agreement with previous reports [28, 48, 49, 33, 29, 35 and 53].

        The results obtained by GC-MS analysis shows that each plant species has a specific quantitative and qualitative composition. The reasons of this variability can be due to different geographical sources, the genotype and the climate; all of this variability influences the chemical composition and the relative concentration of each constituent [54, 55 and 56].

        Also, the drying of leaves influence on composition of the essential oil of Lippia (3). Drying Lippia Citriodora had led to similar results [57] who noted qualitative and quantitative changes occurring in the essential oil of basil during drying in air at 25 ° C. Indeed, after drying for three days, he was a quantitative reduction of the following volatile compounds: 1ol-Octene-3, trans- ocimene, geraniol, geranial, eugenol, neryl acetate, humulene and -muurolene and compounds which increased quantitatively are: -pinene, -pinene, -and -copaene elemen.

    2. Antibacterial activity of the essential oils

    The results of antibacterial activity of the five EOs against The three species of microorganisms are summarized in Table 4 for disk diffusion test, and table 5 for the results of MIC and MBC.

    Table 4: Diameter of inhibition zones in (mm)

    Microorganism species

    Inhibition zone diameter in (mm)

    Mentha (1)

    Mentha (2)

    Lippia (1)

    Lippia (2)

    Lippia (3)

    Positive control

    St. Aureus ATCC 29213

    19 ± 1.73

    15 ± 0

    14.5 ± 0.7

    18.66 ± 1.52

    14 ± 0

    9 ± 0

    E. coli ATCC 25922

    13.66 ± 1.1

    12.33 ± 0.57

    15.33 ± 0.57

    22 ± 2.64

    15 ± 0

    19 ± 0

    Ps. aeruginosa ATCC 27853

    9.5 ± 0.70

    9 ± 1.41

    0 ± 0

    0 ± 0

    0 ± 0

    0 ± 0

    Table 5: MIC and MBC values

    EOs

    St. aureus ATCC 29213

    E. coli ATCC 25922

    Ps. Aeruginosa ATCC 27853

    MIC

    (µl/ml)

    MBC

    (µl/ml)

    MBC/MIC

    MIC

    (µl/ml)

    MBC

    (µl/ml)

    MBC/MIC

    MIC

    (µl/ml)

    MBC

    (µl/ml)

    MBC/MIC

    Mentha (1)

    1.25

    1.25

    1

    1.25

    2.5

    2

    >10

    >10

    Mentha (2)

    1.25

    1.25

    1

    5

    5

    1

    >10

    >10

    Lippia (1)

    0.625

    0.625

    1

    5

    5

    1

    _

    _

    Lippia (2)

    0.625

    1.25

    2

    5

    5

    1

    _

    _

    Lippia (3)

    1.25

    1.25

    1

    5

    5

    1

    _

    _

    By analyzing Table 4, it is seen that the EO Mentha spicata inhibit the growth of three species with a maximum inhibition on St. aureus ATCC 29213, against by the EO of Lippia citriodora, have no effect on Ps. Aeruginosa ATCC 27853, and maximum inhibition for E. coli ATCC 25922. The resistance of Pseudomonas aeruginosa may be due to hydrophobic lipopolysaccharide in the outer membrane which provides protection against different agents [58].

    By a comparison of EO from fresh leaves of plants irrigated by well water (Mentha (1) and Lippia (1)) and those by wastewater (Mentha (2) and Lippia (2)), we see that for Mentha spicata the antibacterial activity decreases when the plant irrigated by wastewater. At the Lippia citriodora, we see the opposite effect, the activity is higher when the plant irrigated by wastewater, however when the EO is extracted from dried leaves, irrigated by wastewater (Lippia (3)), activity that is similar irrigated with well water.

    In Table 5, there is shown the MIC and MBC of 5 EO, it was found that, in most cases, the MIC value was equivalent to the MBC value, indicating a bactericidal effect of the oils tested.

    Generally, the irrigation by urban wastewater did not affect the MBC/MIC ratio, since this ratio values are the same for all of the essential oils.

    The MBC / MIC ratio is used to classify antibiotics according to their characters as bactericides (close to 1) or bacteriostatic (greater than 4) [59]. Our results showed that EO tested has bactericidal activity against

    E. coli ATCC 25922 and st. aureus ATCC 29213.

    The antibacterial activity of EO tested would be result of the presence of citral (neral and geranial), carvone and limonene, known for their antiseptic activity [60]. There is evidence in the literature that the essential oils of some plants of the Lamiaceae family have a moderate to good antibacterial activity [43 and 44].

  4. Conclusion

    In this work, we studied the chemical composition and antibacterial activity of two plants: Mentha spicata and Lippia Citriodora irrigated by well water or wastewater.

    Our study showed that the essential oils from Mentha spicata inhibits the growth of 3 bacterial species with a maximum inhibition for Staphylococcus aureus ATCC 29213, while the EO of Lippia citriodora, has no inhibitory action on Pseudomonas aeruginosa ATCC 27853, and their maximum of inhibition is for Escherichia coli ATCC 25922.

    It is found that fresh leaves of Mentha spicata was the most active of all tested bacteria and the activity of this plant remains effective even when irrigated with wastewater, this is due to the presence of carvone (57,11%) and limonene (27,77%). On the other side, fresh leaves of Lippia Citriodora have a more inhibitory activity when, its irrigated with wastewater.

    Generally, the irrigation by urban wastewater did not affect the MBC/MIC ratio, since this ratio values are the same for all of the essential oils value, indicating a bactericidal effect of the 5oils tested under different conditions.

    This study allowed us to prove the efficacy of OE from these plants against these bacteria. This shows that even these Moroccan flora irrigated by wastewater can be an important subject ofinteresting plant species, including active principles can be used in several areas such as the food and pharmaceutical industries

    ACKNOWLEDGMENTS

    We thank the CNRST (National Center of Scientific and Technical Research, Rabat, Morocco) for GC/MS. We also thank Prof. Angeles Manresa from Unity of Microbiology, Faculty of pharmacy, University of Barcelona, Spain for strains

  5. References

  1. Cowan, M.M., 1999. Plant Products as Antimicrobial Agents. Clin.Microbiol. Rev., 12: 564582

  2. Reische, D.W., D.A. Lillard and R.R. Eitenmiller, 1998. Antioxidants in food lipids In: Ahoh, C.C. and

    D.B. Min (eds.), Chemistry, Nutritionand Biotechnology, pp: 423448. Marcel Dekker, New York

  3. Westh, H., C.S. Zinn and V.T. Rosdahl, 2004. An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microbiol.

    Drug Resist., 10: 169176

  4. Mahady GB (2005). Medicinal plants for the prevention and treatment of bacterial infections. Curr. Pharm. D 11:2405-2427.

  5. Ismaiel A, Pierson MD (1990). Inhibition of growth and germination of C. botulinum 33A, 40 B, 1623, by essential oil of spices. J. Food. Sci. 55:1676-1678.

  6. Bauer K, Garbe D, Surburg H (2001). Common Fragrance and Flavor Materials : Preparations, Properties and uses. 2nd edn. Willey-VHC, Weinheim

  7. Oumzil H, Ghoulami S, Rhajaoui M, Ilidrissi A, Fkih-Tetouani S, Faid M, Benjouad A (2002). Antibacterial and antifungal activity of essential oils of Mentha suaveolens EHRH. Phytother. Res. 16:723-731.

  8. Zenasni L, Bouidida H, Hancali A, Boudhane A, Amzal H, Ilidrissi A, El ouad R, Bakri Y, Benjouad A (2008). The essential oils and antimicrobial activity of four Nepeta species from Morocco. J. Med. Plants Res. 2:111-114.

  9. Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008). Biological effects of essential oils- A review. Food Chem. Toxicol. 46:446-475.

  10. Senatore F (1996). Influence of harvesting time on yield and composition of the essential oil of thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food Chem. 44:1327-1332.

  11. Sara B (2004). Essential oils: Their antibacterial properties and potential applications in foods. A review. Int. J. Food Microbiol. 94:223-253.

  12. Hong EJ, Na Kj, Choi IG, Choi KC, Jeung EB (2004). Antibacterial and antifungal effects of essential oils from coniferous trees. Biol. Pharm. Bull. 27:863- 866.

  13. Bozin B, Mimica-Dukic N, Simin N, Anackov G (2006). Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oil.

    J. Agric. Food. Chem. 54:188-828.

  14. Alzoreky NS, Nakhra K (2003). Antibacterial activity of extracts from some edible plants commonly consumed in Asia. Int. J. Food Microbiol. 80:223-230.

  15. Baser KHC, Demirci B, Demirci F, Koçak S, Akinçi C, Malyer H, Güleryüz G (2002). Composition and antimicrobial activity of the essential oil of Achillea multifida. Planta Med. 68:941-943.

  16. Sylvestre M, Pichette A, longtin A, Nagau F, Legault J (2006). Essential oil analysis and anticancer activity of leaf essential oil of Croton flavens L. from Guadeloupe. J. Ethnopharmacol. 103:99-100.

  17. Bhattacharjee, S.K., 1998. Mentha spicata. In: Handbook of Medicinal Plants. Pointer Publications, Jaipur, India.

  18. Choudhury, R.P., Kumar, A., Garg, A.N., 2006. Analysis of Indian mint (Mentha spicata) of essential, trace and toxic elements and its antioxidant behaviour. J. Pharm. Biomed. Anal. 41 (3), 825832.

  19. Kanatt, S.R., Chander, R., Sharma, A., 2007. Antioxidant potential of mint (Mentha spicata L.) in radiation-processed lamb meat. Food Chem. 100 (2), 451458.

  20. Elmasta, M., Dermirtas, I., Isildak, O., Aboul-Enein, H.Y., 2006. Antioxidant activity of S-carvone isolated from spearmint (Mentha spicata L. Fam. Lamiaceae). J. Liq. Chromatgr. Relat. Technol. 29, 14651475.

  21. Adsersen, A., Gauguin, B., Gudiksen, L., J ¨ager, A.K., 2006. Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitory activity. J. Ethnopharmacol. 104, 418422.

  22. Papachristos, D.P., Stamopoulos, D.C., 2002. Repellent, toxic and reproduction inhibitory effects of

    essential oil vapours on Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). J. Stored Prod. Res. 38, 117 128.

  23. Samarth, R.M., Kumar, A., 2003. Mentha piperita (Linn) leaf extract provides protec-tion against radiation induced chromosomal damage in bone marrow of mice.Indian J. Exp. Biol. 41, 229237

  24. Ozgen, U., Mavi, A., Terzi, Z., Yildirim, A., Coskum, M., Houghton, P.J., 2006. Antiox-idant properties of some medicinal Lamiaceae (Labiatae) species. Pharm. Biol.44, 107112.

  25. Tognolini, M., Barocelli, E., Ballabeni, V., Bruni, R., Bianchi, A., Chiavarini, M., Impiccia-tore, M., 2006. Comparative screening of plant essential oils: phenylpropanoid moiety as basic core for antiplatelet activity. Life Sci. 78, 14191432.

  26. Terblanche´, F.C., Kornelius, G., 1996. Essential oil constituents of the genus Lippia (Verbenaceae) e a literature review. J. Essent. Oil Res. 8, 471e485.

  27. Pascual, M.E., Slowing, K., Carretero, E., S[1]anchez, M.D., Villar, A., 2001. Lippia: traditional uses, chemistry and pharmacology: a review. J.Ethnopharmacol. 76, 201e214.

  28. Carnat, A., Carnat, A.P., Fraisse, D., Lamaison, J.L., 1999. The aromatic and polyphenolic composition of lemon verbena tea. Fitoterapia 70, 44e49.

  29. Santos-Gomes, P.C., Fernandes-Ferreira, M., Vicente, A.M.S., 2005. Composition of the essential oils from flowers and leaves of Vervain (Aloysia triphylla (LHerit.) Britton) grown in Portugal. J. Essent. Oil Res. 17 (1), 73e78.

  30. Valent~ao, P., Andrade, P.B., Areias, F., Ferreres, F., Seabra, R.M., 1999. Analysis of vervain flavonoids by HPLC/Diode array detector method. Its application to quality control. J. Agric. Food Chem. 47, 4579e4582.

  31. Duarte, M.C.T., Figueira, G.M., Sartoratto, A., Rehder, V.L.G., Delarmelina, C., 2005. Anti-Candida activity of Brazilian medicinal plants. J.Ethnopharmacol. 97, 305e311.

  32. Duschatzky, C.B., Martinez, A.N., Almeida, N.V., Bonivardo, S.L., 2004. Nematicidal activity of the essential oils of several Argentina plants against the root- knot nematode. J. Essent. Oil Res. 16 (6), 626e628.

  33. O¨zek, T., Kirimer, N., Baser, K.H.C., Tu¨men, G., 1996. Composition of the essential oil of Aloysia triphylla (LHerit.) Britton grown in Turkey. J. Essent. Oil Res. 8, 581e583.

  34. Ohno, T., Kita, M., Yamaoka, Y., Imamura, S., Yamamoto, T., Mitsufuji, S., Kodama, T., Kashima, K., Imanishi, J., 2003. Antimicrobial activity of essential oils against Helicobacter pylori. Helicobacter 8 (3), 207e215.

  35. Sartoratto, A., Machado, A.L.M., Delarmelina, C., Figueira, G.M., Duarte, M.C.T., Rehder, V.L.G., 2004. Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil. Braz. J. Microbiol. 35, 275e280.

  36. Bensabah F, Lamiri A, Naja J (2013). Effect of purified wastewater from the city of Settat (Morocco) on the quality of spearmint essential oil (Mentha Spicata). ESTIJ, Vol.3, No.1, February 2013.

  37. H. Ismaili, L. Milella, S. Fkih Tetouani, A. Ilidrissi, A. Camporese, S. Sosa, G. Altinier, R.D. Loggia, and R. AAquino, In vivo topical anti-inflamatory and in vitro antioxidant activities of two extracts of thymus satureioides leaves, J. Ethnopharmacol 91(2004), 31 36.

  38. S. Bouhdid, Antimicrobial and antioxidant activities of essential oils: Biotechnological application to improve the quality of natural casings (2009).

  39. Canillac N, Mourey A (1995). Effect of fir an pine essential oil on some ripening microorganisms. M.A.N: Microbiol. Aliments Nutr., 13: 267-273.

  40. Smith-Palmer A, Stewart J, Fyfe L (1998). Antimicrobial properties of plant essential oils and essences five important food-borne pathogens. Lett. Food Microbiol. 26:118-122.

  41. Kokkini, S., Karousou, R., Lanaras, T., 1995. Essential oils of spearmint (carvone-rich) plants from the Island of Crete (Greece). Biochemical Systematics and Ecology 23, 425430

  42. Chauhan, R.S., Kaul, M.K., Shani, A.K., Kumar, A., Rama, G., Tawa, A., 2009. Chemical composition of essential oils in Mentha spicata L. accession [III(J)26] from North-West Himalayan region, India. Industrial Crops and Products 29, 654656.

  43. arer, E., Toprak, S.Y., Otlu, B., Durmaz, R., 2011. Composition and antimicrobial activity of the essential oil from Mentha spicata L subsp. spicata. Journal of Essential Oil Research 23, 106108.

  44. Sokovic, M., Van Griensven, L.J.L.D., 2006. Antimicrobial activity of essential oils and their components against the three major pathogens of the cultivated button mushroom Agaricus bisporus. European Journal of Plant Pathology 116, 211224.

  45. Bellakhdar, J., Il Idrissi, A., Canigueral, S., Iglesias, J., Vila, R., 1994. Composition of lemon verbena (Aloysia triphylla (LHerit.) Britton) oil of Moroccan origin. J. Essent. Oil Res. 6, 523e526.

  46. Catalan, C.A.N., de Lampasona, M.E.P., 2002. The chemistry of the genus Lippia (Verbenaceae). In: Kintzios, S.E. (Ed.), Oregano: The genera Origanum and Lippia, first ed. Taylor and Francis Inc., pp. 127e149.

  47. Crabas, N., Marongiu, B., Piras, A., Pivetta, T., Porcedda, S., 2003. Extraction, separation and isolation of volatiles and dyes from Calendula officinalis L. and Aloysia triphylla (LHer.) Britton by supercritical CO2.

    J. Essent. Oil Res. 15 (5), 350e355.

  48. Kim, N.-S., Lee, D.-S., 2004. Headspace solid-phase microextraction for characterization of fragrances of lemon verbena (Aloysia triphylla) by gas chromatographyemass spectrometry. J. Sep. Sci. 27, 96e100.

  49. Montes, M., Valenzuela, L., Wilkomirsky, T., Arrive´, M., 1973. Sur la composition de l essence d Aloysia triphylla (cedro´n). Planta Med. 23, 119e124.

  50. Velasco-Negueruela, A., Pe´rez-Alonso, M., Guzm[1]an, C.A., Zygadlo, J.A., Ariza-Espinar, L., Sanz, J., Garc´a-Vallejo, M.C., 1993. Volatile con-stituents of four Lippia species from Co´rdoba (Argentina). J. Essent. Oil Res. 5, 513e524.

  51. Von Kaiser, R., Lamparsky, D., 1976b. Caryophyllane-2,6-b-oxide, a new sesquiterpenoid compound from the oil of Lippia citriodora KUNTH. Constituents of Verbena oil, 2nd communication. Helv. Chim. Acta 59, 1803e1808.

  52. Zygadlo, J.A., Lamarque, A.L., Maestri, D.M., Guzm[1]an, C.A., Lucini, E.I., Grosso, N.R., Ariza- Espinar, L., 1994. Volatile constituents of Aloysia triphylla (LHerit.) Britton. J. Essent. Oil Res. 6, 407e409.

  53. Scorzoni. L, Bendaducci, T. Fusco, A.M. Almeida,

    D.H. Siqueira Silva, V.S. Bolzani and MJS. Mendes Gianinnil, 2007; The use of standard methodology for

    determinations of Antifongal activity of natural products against medical yeast Candila sp. and Cryptococcus SP. Brazilian J. Microbiol., 38: 391-397;

  54. Masotti V, Juteau F, Bessière JM, Viano J (2003). Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J. Agric. Food Chem. 51:7115-7121.

  55. Angioni A, Barra A, Coroneo V, Dessi S, Cabras P (2006). Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas

L. ssp stoechas essential oils from stem/leaves and flowers. J. Agric. Food Chem. 54:4364-4370.

Leave a Reply