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M. Sienkiewicz, P. Denys, E. Kowalczyk: Antibacterial and immunostimulatory effect of essential oils

Antibacterial and immunostimulatory effect of essential oils

Monika Sienkiewicz1, Pawe Denys2, Edward Kowalczyk3
Medical University of Lodz, Poland: 1Department of Medical and Sanitary Microbiology; 2 Orthopedic Clinic; 3Pharmacology and Toxicology Department

SUMMARY
Antibacterial and immunostimulatory effect of essential oils Sienkiewicz M.1, Denys P.2, Kowalczyk E.3 Medical University of Lodz, Poland: 1Department of Medical and Sanitary Microbiology; 2Orthopedic Clinic; 3Pharmacology and Toxicology Department Int. Rev. Allergol. Clin. Immunol., 2011; Vol. 17, No. 1-2

STRESZCZENIE
Dziaanie przeciwbakteryjne i immunostymulujce olejkw eterycznych Sienkiewicz M.1, Denys P.2, Kowalczyk E.3 Uniwersytet Medyczny w odzi: 1Zakad Mikrobiologii Lekarskiej i Sanitarnej; 2Klinika Ortopedii; 3Zakad Farmakologii i Toksykologii Int. Rev. Allergol. Clin. Immunol., 2011; Vol. 17, No. 1-2

Volatile or essential oils aetherolea are secondary plant metabolites containing different compounds. The oils have wide therapeutic action. Strong antiseptic activity of essential oils has been known all over the world for many centuries. A large number of essential oils and their constituents have been investigated for their microbial properties against some bacteria, fungi, viruses and protozoa. G-positive bacteria are generally more susceptible to essential oils than G-negative ones. So far there been no reports on growing resistance of bacterial strains to oils. The antimicrobial and immunostimulatory activity of essential oils is strictly connected to their chemical composition. Essential oils of thyme, oregano, cinnamon, tea tree, eucalyptus, lavender, salvia and mint, were found to possess the strongest antibacterial and antifungal properties. Essential oils show high activity against even in the methycillin-resistant staphylococci, vancomycin-resistant streptococci and against Gram-negative ones. Thyme and eucalyptus oils have been the most inhibiting against respiratory tract pathogens. Oregano oil appears to have a wide spectrum of antibiotic activity against microflora and this is recommended in fighting intrahospital infections. The article clearly demonstrate that essential oils presents a great potential for medical procedures and for pharmaceutical industries. Key words: antimicrobial activity, pharmacological properties

Olejki eteryczne s produktami metabolizmu wtrnego rolin. Maj one wielokierunkowe oddziaywanie terapeutyczne. Silne waciwoci antyseptyczne olejkw eterycznych znane s na caym wiecie od wielu wiekw. Olejki eteryczne i ich skadniki dziaaj przeciwbakteryjnie, przeciwgrzybowo, przeciwwirusowo i przeciwpierwotniakowo. Bakterie Gram-dodatnie s bardziej wraliwe na dziaanie olejkw eterycznych ni bakterie Gram-ujemne. Do tej pory nie odnotowano doniesie o rosncej opornoci szczepw bakteryjnych na dziaanie olejkw eterycznych. Dziaanie przeciwdrobnoustrojowe i immunostymulujce olejkw eterycznych jest cile zwizane z ich skadem chemicznym. Do najbardziej skutecznych olejw nale m in. olejki pozyskane z: tymianku, oregano, cynamonowca, drzewa herbacianego, eukaliptusa, lawendy, szawi i mity. Wykazuj one silne waciwoci bjcze nawet wobec metycylinoopornych gronkowcw, wankomycynoopornych paciorkowcw, rwnie wobec bakterii Gram-ujemnych. Olejek tymiankowy i eukaliptusowy stosowane s w zwalczaniu infekcji ukadu oddechowego. Olejek oreganowy ze wzgldu na szerokie spektrum aktywnoci moe by polecany w zwalczaniu patogenw odpowiedzialnych za zakaenia szpitalne. Artyku ten jasno ukazuje przydatno olejkw eterycznych w lecznictwie i obrazuje ich szeroki potencja terapeutyczny. Sowa kluczowe: waciwoci przeciwdrobnoustrojowe, dziaanie terapeutyczne

The increasing resistance of many clinical strains of bacteria to commonly used antibiotics and chemotherapeutics in pharmacological practice are looking for safe and effective factors that could be used to treat persistent bacterial infections [4]. Several research centers have been interested in essential oils. Essential oils are a very differential group of plant se-

condary metabolites which have long been used in aromatherapy, dermatology and cosmetology [17]. Their wide and complex effects, synergy of action also in combination with antibiotic therapy and the no reports on the acquisition of resistance mechanisms of bacteria to their constituents make them a valued complement to therapy in human infectious diseases [31].

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Oils are produced and stored in plant secretionexrection tissue both endogenous (including oils, oils reservoirs, secretion canals) and exogenous (sting glands, as in the plants from Lamiaceae and Asteraceae family). Components of essential oils are synthesized through a series of biochemical reactions and are products of side biosynthesis pathways. The composition of essential oil depends on the family, genus, species and chemotype of a plant, as well as from the material which it is obtained, growing conditions, harvesting season and geographical origin. Chemically, oils are mixtures containing from several dozen to several hundred compounds. They mainly contain terpenics derived from pirophosphorane isopentyl (terpenic essential oils), phenylpropanoids and the aminoacid phenylalanine derivatives (nonterpenic essential oils). Compounds in these groups are hydrocarbons, alcohols, aldehydes, ketones, esters and ethers. Sulphurous compounds (mustard essential oils), nitrogen, acetylene derivatives (poliins), tropolons, coumarins, organic acids may also be components of essential oils [2, 5, 20]. About 300 essential oils are produced on industrial scale in the world. The most important essential oils are: peppermint, cornmint and spearmint and citrus such as: lemon, orange, lime, bergamot, grapefruit, and also rose, lavender and eucalyptus. Oils are obtained from plant material by steam distillation or by mechanical pressing of the peel of citrus fruit or a dry distillation method. Terms of naming oils and raw materials from which they originate, the determination of the crop from which they are received, as well as methods for their examination and determination of quality parameters are governed by the relevant provisions.

Mill. (Lamiaceae), Salvia officinalis L. (Lamiaceae), Rosmarinus officinalis L. (Lamiaceae) [26]. The mechanism of action of essential oils may be related to their lypophilic character. They are well absorbed by the nasal, oral, gastric, intestinal mycosis membranes and skin. In large concentrations they may have irritant activity. The active compounds contained in the oils are incorporated into cell membranes and influence enzyme and ion channel function and receptor proteins. Oils do not accumulated in the human body and are neutralized by binding to glucuronic acid and eliminated with urine [5]. Essential oils demonstrate bactericidal activity against both Gram-positive bacteria as well as however, lover due to complex structure of cell walls against Gram-negative bacteria with the range from 50 to 1000 mg/ml and even at 0,25-1.0 mg/ml concentrations for most active oils. Antimicrobial efficacy of essential oils against bacteria, fungi, protozoa and viruses is strictly linked to their chemical composition. Bactericidal properties depend on the lipophilic character of hydrocarbon skeleton and hydrophilic character of functional groups. The highest antibacterial activity and the widest spectrum of action are demonstrated by phenolic compounds such as: carvacrol, thymol, eugenol. There are even 20 times stronger than pure phenol. In addition, such strong bactericidal properties are show by the oil extracted from the aldehyde containing bark of cinnamon. The second group of biologically active essential oils are oils with alcohols: terpinen-4-ol, a-terpineol, geraniol, cytronellol, menthol, linalol. Antimicrobial properties of oils are also associated with ketones: thujene, camphor, menthone, carvone, and ethers: anethole, cyneole.

THERAPEUTIC EFFECT
Essential oils have a range of therapeutic effects for instance: anti-inflammatory antiphlogistica (Chamomollae oleum, Millefolii oleum), anaesthetic anaestetica (Melissae oleum, Caryphylii oleum, Citri oleum, Lavandulae oleum), cholagogic and cholepoietic cholagoga, choloerectica (Hyperici oleum, Chelidonii oleum, Menthae piperitae oleum, Raphanus oleum), spasmolytic and relaxing spasmolytica (Foeniculi oleum, Carvi oleum, Coriandrii oleum), diuretic diureticum (Juniperi oleum, Petroselini oleum), expectorant expectorantia (Pini oleum, Thymi oleum, Serpylli oleum, Eucalipti oleum). Oils in particularly valuable medicinal properties, mainly due to antibacterial, antifungal and antiviral activities are extracted from Melaleuca alternifolia (M. and B.) Ch.) (Myrtaceae), Thymus vulgaris L. (Lamiaceae), Eugenia caryphyllata Thun. (Myrtaceae), Lavandula angustifolia

ESSENTIAL OILS CONTAINING PHENOLS

Thymol and carvacrol occur in different proportions in essential oils produced by plants from Lamiaceae family. Thyme (Thymus vulgaris L.), oregano (Origanum vulgare L.) and savory (Satureja hortensis L.) are of particular importance [18, 26]. Inhalation of the essential oil of thyme are used in the treatment of persistent respiratory tract infection. Thyme oil shows growth inhibiting activity of bacterial strains: Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, Kliebsiella pneumoniae, Salmonella typhimurium, Mycobacterium smegmatis, Pseudomonas aeruginosa, Clostridium botulinum, Clostridium perfringens, Bacillus subtilis genera [18]. Due to thymol and carvacrol, bactericidal activity has also been obtained from

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M. Sienkiewicz, P. Denys, E. Kowalczyk: Antibacterial and immunostimulatory effect of essential oils

other species of thyme such as Thymus zygis L., T. serpyllum L., T. kotschyanus Boiss. & HoH., T. persisus L., T. longicaulis C. Presl [34, 28]. Subject literature presents both the antimicrobial properties of thyme essential oils and oregano and also their antioxidant activity. Chemotypes of thyme oils extracted from: Thymus munybyanus De Noe, T. pallescens, T. numidicus Poiret, T. guyonii De Noe and origanum: Origanum glandulosum Desf. and O. floribundum Munby. containing the largest amount of thymol, a-carvacrol and terpineol showed the strongest antioxidant and inhibitory properties against the standard strains of Listeria monocytogenes [14]. Antibacterial activity of essential oil obtained from Origanum scabrum Boiss. & Heldr. against the standard strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Enterobacter cloace, Klebsiella pneumoniae is mainly dependent on the carvacrol content. The essential oil obtained from Origanum microphyllum Vogel containing alcohols terpin-4-ol, linalool and g-terpinene as dominant components shows less antimicrobial properties [1]. Studies of biological properties of savory oil (Satureja hortensis L.) containing phenols as predominant compounds, showed their antioxidant and antimicrobial properties against standard strains of both Gram-positive and Gramnegative bacteria [13]. Biological influence of essential oils obtained from Ocimum basilicum L., Origanum vulgare L. and Thymus vulgaris L. is strictly related to the content of phenolic compounds. Basilic oil containing alcohols, mainly linalool and estragole shows less activity against standard strains of bacteria: Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis, Shigella sonnei, Micrococcus flavus, Sarcina lutea, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis than savory and thyme essential oils [3].

coccus faecalis, Streptococcus pyogenes, Bacillus cereus, B. subtilis, B. brevis), and Gram-negative (Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis, S. typhi, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa) due to the high content of eugenol [23, 25].

ESSENTIAL OILS CONTAINING NO PHENOLIC COMPOUNDS

Oils containing no phenolic compounds with high antimicrobial activity include: tea tree oil, eucalyptus oil, lavender, rose and sandalwood oil. Tea tree oil is mainly obtained from leaves and twigs of the Melaleuca alternifolia (M. and B.) Ch., as well as from two other species: M. linariifolia Smith and M. dissitiflora F. Muell. Cajeput oil is obtained for M. leucadendron L., while niaouli oil from M. quinquenervia (Cav.) ST Blake. Chemotype with low cineol content is of highest practical use out of the several chemotypes of the tea tree oil. Monoterpenic alcohol terpinen-4-ol (29-45%) is predominant component of this essential oil, in smaller quantities it contains: g-terpinene, a-terpinene, a-terpineol, 1.8-cyneol, p-cymene, a-pinene and limonene. Tea tree oil is considered as the strongest aromatherapeutic antimicrobial agent. It is used in the treatment of respiratory diseases, persistent gynecological infections, and dermatology inter alia [7, 21]. Tea tree oil strongly inhibits the proliferation of Gram-positive and Gram-negative bacteria, including those particularly resistant to antibiotics such as Pseudomonas aeruginosa. It is active against aerobic and anaerobic bacteria. Literature reports inhibiting activity of the oil against bacteria isolated from the skin surface, such as staphylococcus: Staphylococcus aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, S. warneri, S. xylosus, and micrococci: Micrococcus luteus, M. varianus and Corynebacterium sp. Gram-negative bacteria isolated from skin (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumanii) showed sensitivity to this oil however, in slightly higher concentrations [7]. Studies on antimicrobial properties of tea tree oil against bacteria isolated from chronic urinary tract infections show that the oil inhibits growth of Gram-positive bacteria, such as: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Corynebacterium ulcerans, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans and Gram-negative ones, such as Escherichia coli, Enterobacter georginosa, Pseudomonas aeruginosa, Acinetobacter calcoaceticus var. Lwoffi, Xanthomonas maltophilia, Haemophilus parainfluenzae, Neisseria

ESSENTIAL OILS CONTAINING EUGENOL

Phenylpropanoid eugenol belongs to the phenolic compounds and also shows strong antimicrobial properties. Plants which provide essential oil containing up to 90% of this compound include: clove (Syzygium aromaticum, syn. Eugenia caryphyllata Thun.) cinnamon leaves (Cinnamomum zeylanicum Blume), bay (Laurus nobilis L.), numeg (Myristica fragilis Hott.) and a few species basil such as: Ocimum basilicum L., O. gratissimum L. [18]. Research shows that the oil obtained from cinnamon leaves and clove strongly inhibits the growth of standard strains of bacteria Gram-positive (Staphylococcus aureus, Listeria monocytogenes, Entero-

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mucosa, Neisseria polysaccharea, Branhamella catharalis. The results of the conducted studies present that the oil is used in 1:200 dilution inhibits the growth of all bacteria isolated from the upper respiratory tract [15]. Clinical studies using the tea tree and lavender oils demonstrated not only a strong antibacterial activity of the tested oils but also their about its immunostimulatory effect. After inhalation of essential oils absence of bacteria in sputum, alleviation of clinical symptoms, increased activity of chemiluminescence of granulocytes, as well as reducing the incidence of infection in patients were observed with bacterial respiratory infections [33]. Lavender oil also shows strong antiseptic activity. It is used in the treatment of mouth, throat, upper respiratory tract and lungs infections and in dermatology in the treatment of ulcers, burns and difficult to cicatrize wounds. The main components of the oil are linalol and linalil acetate. Oil of lavender (Lavandula angustifolia Mill.) has strong antiseptic activity against methicillin resistant strains of Staphylococcus aureus (MRSA) and against vancomicin resistant strains of bacteria of the Enterococcus sp genus (VRE) [8, 27]. According to the literature this oil proved to be helpful in the fighting against respiratory infections caused by erythromycin, amoxicillin and cefalotin resistant strains of Haemophilus influenzae [10]. The oil obtained from Lavandula heterophylla Avonview shows growth inhibitory activity against: Streptococcus pyogenes, Enterobacter aerogenes, Staphylococcus aureus, MRSA, Pseudomonas aeruginosa, Citrobacter freundii, Proteus vulgaris, Escherichia coli, VRE, Shigella sonnei, and Propionibacterium acnes [8,12]. Due to the high content of menthol and menton mint essential oils have also strong antibacterial properties. They are mainly obtained from peppermint (Mentha piperita (L.) Hudson peppermint), field mint (M. arvensis L. cornmint) and green mint (M. spicata L. spermint) [18]. The main components of mint oils are: 1-menthol (20-54%), 1-menton (15-43%), mentyl acetate (1-29%), menthofuran (1-8%). Oil used for medicinal purposes should contain: 44% of menthol and its isomers, 4,5-10% menthol esters, 15-32% of ketones (mentone, izomentone), small amounts of mentofurane, terpens and their oxides are acceptable. Oil from various species of mint has antimicrobial properties inter alia against: Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Clostridium perfringens, Bacillus brevis, Vibrio cholerae. Peppermint oil may be used to treat severe infections of the digestive tract caused by Salmonella enteritidis, S. typhimurium, Shigella sonnei, Listeria monocytogenes and Escherichia coli. It shows strong also show inhibiting activity against

methicillin resistant strains of Staphylococcus aureus and against Helicobacter pylori genera [18, 32]. The content of biologically active ketones and alcohols affect the antimicrobial properties in the Salvia sp. Two species of sage (Salvia officinalis L.) and (Salvia sclarea L.) are of particular therapeutic use. These essential oils have a different composition, in Salvia officinalis L. the predominant components of essential oil are: a-tujon (2-46%), b-thujene (2,5-30%), camphor (2-46%), 1.8-cyneol (2-18%) while in Salvia sclarea L.: linalil acetate (0,5-74%), linalol (1-29%), geraniol (1-15%), neral (0-12%), nerol (0,5-8%), germacrene D (2-4%) [16, 19]. Studies on antimicrobial properties of essential oil of sage and rosmarinic oil (Rosmarinus officinalis L.) shown similar activity against the standard bacterial strains: Staphylococcus aureus, Streptococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, due to the content of thujene, camphor, and 1.8-cyneol eucalyptol [29, 23]. Eucalyptol oil used in respiratory diseases has strong bactericidal activity and also antiinflammatory, expectorant and stimulating secretions of bronchial challenge properties. Oil obtained from the leaves of eucalyptus (Eucalyptus globulus Labill.) contains 1.8-cyneol from 70 to 95% while isolated from fruits, buds and shoots from 15 to 75% of this compound [9]. This oil has growth inhibiting activity against bacterial strains which have been isolated from respiratory infections such as: Staphylococcus aureus, Streptococcus pyogenes, S. pneumoniae and Haemophilus influenzae, H. parainfluenzae, Stenotrophomonas maltophilia [30, 6, 11]. It has shown high effectiveness against anaerobic bacteria which have been isolated from oral and respiratory infections diseases. In low concentrations it inhibits the growth of Gram-negative bacilli of the Prevotella sp, Bacterioides sp, Fusobacterium sp, genus and gram-positive cocci of the Anerococcus and Micromonas sp genus. It may also be useful in the treatment of urinary infections caused by clinical strains of Escherichia coli. Research has shown sensitivity of strains isolated from urine and resistant to commonly used antibiotics to eucalyptus and rosmarinic oils [24]. Rich in hydrocarbons essential oils obtained from coniferous trees, pine oil (Pini Oleum) and oil of fir (Abies Oleum) demonstrate relatively less antimicrobial activity [22].

CONCLUSIONS
Phytochemical and pharmacological research rapidly increases the list of plant resources favorably affecting human body. Essential oils, a diverse group of plant metabolites, seem to be interesting. They have long been used in aromatherapy, dermatology and

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M. Sienkiewicz, P. Denys, E. Kowalczyk: Antibacterial and immunostimulatory effect of essential oils

cosmetics. Their broad and complex activity, synergy of action, also in combination with antibiotic therapy and the lack of reports about emergence of resistance mechanisms of bacteria to their constituents makes them a valued complement to infection therapy in human diseases.

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Correspondence: Monika Sienkiewicz MD Department of Medical and Sanitary Microbiology, Medical University in Lodz, Poland 90-647 Lodz, Hallera Sq. 1, 90-647, phone/fax: 48 42 639 31 98 e-mail: monika.sienkiewicz@umed.lodz.pl