Antibacterial activity of essential oils, hydrosols and plant extracts from Australian grown Lavandula spp.

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1 The International Journal of Aromatherapy (2006) 16, 9 14 The International Journal of Aromatherapy intl.elsevierhealth.com/journals/ijar Antibacterial activity of essential oils, hydrosols and plant extracts from Australian grown Lavandula spp. T. Moon, J.M. Wilkinson *, H.M.A. Cavanagh School of Biomedical Sciences, Charles Sturt University, Locked bag 588, Wagga Wagga, NSW 2678, Australia KEYWORDS Antibacterial; Lavender; Lavandula; Essential oil; Hydrosol Summary Although there is considerable anecdotal information about the antibacterial activity of lavender oils, much of this has not been substantiated by scientific or clinical evidence. In this study we assessed the activity of lavender essential oils, hydrosols and aqueous and ethanolic foliage extracts from a range of Australian grown Lavandula species. The results support the anecdotal use of lavender oils as antibacterial agents and demonstrated that some oils which had previously not been investigated (e.g., Lavandula heterophylla) display good antibacterial activity against a range of bacteria including Streptococcus pyogenes, Staphylococcus aureus, MRSA, Citrobacter freundii, Proteus vulgaris, Escherichia coli, VRE and Propionibacterium acnes. Pseudomonas aeruginosa was the only bacterium not susceptible to any essential oil. There was considerable variability in the activity of the essential oils however; no one oil produced the highest level of antibacterial activity against all bacteria. No correlation was observed between the percentage of major chemical components and antibacterial activity. The lavender hydrosols and aqueous foliage extracts did not have any antibacterial activity. Six of the ethanolic extracts displayed activity against Pr. vulgaris but no activity against any other organism. Further work is required to determine whether these in vitro results will be realised in a clinical environment but it is clear that not all lavenders are equal in terms of their antibacterial properties. c 2006 Elsevier Ltd. All rights reserved. Introduction * Corresponding author. Tel.: ; fax: address: jwilkinson@csu.edu.au (J.M. Wilkinson). Within the scientific and medical literature lavender essential oils have been investigated for a number of properties, for example, Lavandula multifida extracts have demonstrated anti-inflammatory /$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi: /j.ijat

2 10 T. Moon et al. activity (Sosa et al., 2005) while others have reported positive effects on mood following inhalation of lavender oils or fragrance (Field et al., 2005; Morris, 2002). However, perhaps the bestknown use of lavender is as an antiseptic or antibacterial agent. Lavender essential oils are advocated for their use as an antibacterial agent in both early and modern aromatherapy texts (Gattefosse, 1995; Lawless, 1992). Gattefossé, for example, in his 1937 aromatherapy text describes the use of essences of lavender essential oils as an antiseptic mouthwash and in embalming (Gattefosse, 1995). Hammer et al. (1999) included two L. angustifolia oils (identified by the authors as French lavender and Tasmanian lavender) in their study of a range of essential oils and found that both oils exhibited antibacterial activity with minimum inhibitory concentrations (MIC) in the range of 0.5% to >2% (v/v), with little difference between the two oils. Dadalioğlu and Everendiliek (2004) showed that L. stoechas (Spanish lavender) had a strong antibacterial action against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus. Similarly Lis-Balchin and Deans (1997) and Lis-Balchin et al. (1998) showed that lavandin, French lavender, spike lavender, Bulgarian lavender and generic lavender (type unspecified) essential oils all have activity against a large number of bacteria and fungi. For example, Bulgarian lavender essential oil inhibited 23 of 25 different bacteria while lavandin inhibited 17 of 25 bacteria. Unfortunately, although in both studies the authors did GC/ MS analysis of the oils, they did not seek to verify the plant source of each of the lavender oils, relying only on common names. These studies suggest that the anecdotal use of lavender as an antibacterial agent may be justified and that products, for example toiletries and cosmetics, incorporating these essential oils may have some additional value as therapeutic products. In Australia the best-known lavender essential oil is that from Bridestowe Lavender in Tasmania. This essential oils is distilled from L. angustifolia (also known as True or English lavender); however a study of the Australian lavender industry (Peterson, 2002) has demonstrated that most lavender growers grow several varieties (e.g., L. x intermedia (lavandin), L. stoechas and L. x allardii), often depending on favorability of local growing condition. Unlike L. angustifolia essential oil which has value as a perfumery product these other lavender essential oils are often incorporated into products such as shampoos, soaps, lip balms and other cleansing products, or the flowers are dried for use a pot-pourri. In addition, the by-products of distillation (the hydrosol) are gaining popularity as a cosmetic and therapeutic product in their own right (Catty, 2001). In this study we examined the antibacterial activity of a range of Australian grown lavender essential oils, hydrosols and aqueous and ethanolic extracts. Materials and methods Essential oils and lavender extracts A range of lavender essential oils, and some hydrosols, were obtained directly from growers/distillers or purchased commercially. All but one of the L. angustifolia oils were distilled from Australian grown plants. A complete list of the plants used and the products tested in this study are listed in Table 1. Samples of fresh foliage were obtained from the lavender collection at Charles Sturt University for production of aqueous and ethanolic extracts. Flowers heads were removed and the foliage dried at room temperature for one week. The dried foliage was then ground to a fine powder using a commercial coffee/spice grinder (grinding time s). The powder was then sealed in sterile glass jars and stored at room temperature in the dark until required. Ethanol extracts were prepared by shaking 5 g foliage powder in 50 ml ethanol for 16 h at 45 C and 160 rpm. The extract was cooled and filtered though Advantec #1 filter paper to remove particulate matter and freeze-dried in 100 ml Quickfit flasks attached to a rotary evaporator. The resulting solid was removed from the flask using a sterile spatula and a minimum volume of ethanol (approximately 12 the weight of sample). Each sample was centrifuged at 2000 rpm for 3 min and the supernatant used in the disc diffusion assay. Aqueous extracts were prepared by boiling, in a covered beaker, 5 g foliage powder in 250 ml sterile nanopure water for 5 min. The cooled extract was then filtered using Advantec #1 filter paper and a 0.45 lm membrane filter (Sartorius, Australia). The resulting extract was stored at 20 C until required. Microorganisms Lavender essential oils and hydrosols were assayed against the following microorganisms: Gram positive bacteria Enterobacter aerogenes, Propionibacterium acnes, Staph. aureus, MRSA, Streptococcus pyogenes & VRE; Gram negative bacteria Citrobacter freundii, Escherichia coli, Proteus vulgaris,

3 Antibacterial activity of Australian grown Lavandula spp. 11 Table 1 Source of lavender products used in this study Lavandula spp. Product tested Main constituents a L. angustifolia (UK) Essential oil 41% Linalyl acetate; 29% linalool L. angustifolia Essential oil 40% Linalyl acetate; 36% linalool L. angustifolia Egerton Blue Foliage L. angustifolia Avice Hill Foliage L. x allardii Essential oil (single-distilled and double-distilled oils), hydrosol, foliage Single distilled: 39% 1,8-cineole; 13% camphor. Double distilled: 34% 1,8-cineole; 17% camphor. Hydrosol: 39% camphor L. x intermedia Grosso Essential oil, hydrosol, foliage Oil: 24% linalyl acetate; 34% linalool L. x intermedia Seal Essential oil, hydrosol, foliage Oil: 36% linalool; 15% 1,8-cineole L. x intermedia Miss Donnington Essential oil, hydrosol, foliage Oil: 20% camphor; 12% linalool; 12% 1,8-cineole. Hydrosol: 20% linalool; 17% camphor L. x heterophylla Essential oil, foliage Oil: 40% linalool; 18% camphor L. stoechas Avonview Essential oil, foliage Oil: 48% camphor; 22% fenchone a GC/MS data was not available for all samples tested. Pseudomonas aeruginosa, Shigella sonnei; and the yeast Candida albicans. Aqueous and ethanolic extracts were assayed against all of the above except P. acnes. Organisms were originally obtained from the culture collection of the University of New South Wales, Australia. The bacteria were grown in nutrient broth (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, water to 1 L) and/or nutrient agar (as for nutrient broth plus 15 g/l agar). Normal bacterial growth was evaluated by growth on nutrient agar at appropriate conditions (37 C, aerobic or anaerobic as required) in the absence of test substance. Anaerobic conditions were achieved by placing agar plates in an anaerobic jar (AnaeroGenä, Oxoid, UK). Antibacterial assay Antibacterial activity was determined using the disc diffusion assay. Overnight (18 h; 37 C with shaking 120 rpm) broth cultures of bacteria were freshly prepared for each assay. Agar plates (20 ml) were prepared, allowed to set, and then surface dried (37 C, 30 min). Broth cultures were vortexed for 30 s and 500 ll immediately removed and spread over the surface of the agar and then surface dried (37 C, 15 min). 10 ll of essential oil, hydrosol or extract was pipetted onto a 6 mm sterile disc (Oxoid) and the disc placed onto the surface of the prepared agar plate. Following 24 h incubation at 37 C the diameters of the zone of inhibition for each dilution were then recorded in mm (including disc). For control plates 10 ll nanopure water was pipetted onto the disc. Assays were completed in triplicate (i.e., 3 discs per plate) and assays repeated independently three times. Assays were also repeated using commercial antibiotics (Oxoid antibiotic discs) as positive controls (Table 2). Differences between size of zones of inhibition were tested using Students t test or ANOVA (with Tukey post-test) with differences being deemed significant where p < Results The results of the disc diffusion assay of nine lavender essential oils against 11 bacteria and the yeast C. albicans are shown in Table 3. There was considerable variability in the size of zone of inhibition with the different oils and range of susceptible organisms for any single oil. Ps. aeruginosa was the only bacterium that was not susceptible to the oils; the small zone (6.4 ± 0.9 mm) produced with European L. angustifolia oil was not significantly different to the other oils. Overall, no essential oil was observed to be the best against all organisms, with Strep. pyogenes, Pr. vulgaris and C. albicans having consistently larger zones than other organisms. There were few differences in the susceptibility of organisms to the two L. angustifolia oil samples, however where statistically significant differences existed the European oil produced the greater inhibition. For example, C. freundii was susceptible to the European sample while the Australian oil had no effect. Other organisms for which differences were observed were P. acnes (p = 0.011), Pr. vulgaris (p = 0.001), Sh. sonnei (p < 0.001) and Staph. aureus (p < 0.001). Similarly few statistically

4 12 T. Moon et al. Table 2 Antibiotic sensitivity assay used as positive controls Size of zone of inhibition (mm) (mean ± standard deviation) Tetracycline (30 lg) Methicillin (10 lg) Ampicillin (10 lg) Vancomycin (30 lg) Naladixic acid (30 lg) Citrobacter freundii No zone No zone 12.3 ± ± ± 0.6 Enterobacter aerogenes 15.2 ± 0.5 No zone No zone No zone 15.1 ± 0.5 Escherichia coli 19.0 ± 0.7 No zone 11.6 ± 0.8 No zone 15.9 ± 0.3 MRSA 12.8 ± 0.2 No zone 7.4 ± ± 0.4 No zone Proprionibacterium acnes 24.3 ± ± ± ± 0.4 No zone Proteus vulgaris 17.9 ± 0.7 No zone No zone No zone 18.9 ± 0.4 Pseudomonas aeruginosa 8.7 ± 0.5 No zone No zone No zone No zone Shigella sonnei 17.5 ± 0.1 No zone 17.1 ± 0.3 No zone 18.3 ± 0.2 Staphylococcus aureus 21.5 ± ± ± ± ± 0.6 Streptococcus pyogenes 16.5 ± ± ± ± 0.7 No zone VRE 11.1 ± 0.3 No zone No zone 21 ± 1.7 No zone Candida albicans No zone No zone No zone No zone No zone significant differences were observed between the double and single-distilled L. x allardii oils; E. aerogenes (p = 0.009), E. coli (p = 0.004) and Pr. vulgaris (p = 0.006). Statistically significant differences in antibacterial activity observed between the three L. x intermedia samples for C. freundii (p = 0.009), E. aerogenes (p < ), E. coli (p < ), Pr. Vulgaris (p = 0.003), Sh. Sonnei (p = 0.002) and Staph. aureus (p < ). Apart from Staph. aureus, the zone of inhibition for L. x intermedia Miss Donnington and Seal were significant larger than that for Grosso. However Miss Donnington did not consistently produce greater zones of inhibition than Seal, or vice versa. For C. freundii and Pr. vulgaris there was no difference between these oils, while for E. aerogenes, E. coli and Sh. sonnei Seal produced significantly larger zones of inhibition that Miss Donnington. For Staph aureus Miss Donnington produced significantly larger zones of inhibition than both Grosso and Seal. In contrast, no antibacterial activity was observed with either the hydrosols or with the aqueous extracts. The only organism susceptible to the ethanolic extracts of the lavender foliage was Pr. vulgaris: L. angustifolia Avice Hill (8.2 ± 0.3 mm), L. x allardii (8.3 ± 0.2 mm), L. angustifolia Egerton Blue (7.6 ± 0.3 mm), L. x heterophylla (8.4 ± 0.5 mm), L. x intermedia Miss Donnington (7.4 ± 0.2 mm) and L. stoechas Avonview (9.2 ± 0.5 mm). Small zones of inhibitions were also recorded with the ethanolic extracts of L. stoechas Avonview against C. albicans (6.2 ± 0.3 mm) and Strep. pyogenes (6.4 ± 0.5 mm) and with the extract of L. x intermedia Miss Donnington against Strep. pyogenes (6.5 ± 0.5 mm); however these zones are unlikely to be clinically significant. Discussion The traditional and anecdotal use of lavender essential oils for their antibacterial activity has been substantiated by this study. We have shown that, with the exception of Ps. aeruginosa, all bacteria tested and C. albicans were susceptible to the lavender essential oils. As the bacteria used in this study included common human pathogens and organisms that resistant to conventional antibiotics (e.g., VRE and MRSA) these results open the opportunity for the use of lavender essential oils as an antibacterial ingredient in toiletries, cosmetics and cleaning products. No oil proved to be consistently superior to the rest, and there was no preferential activity against Gram positive versus Gram negative organisms. Pr. vulgaris and Strep. pyogenes were found to be most susceptible of the bacteria tested. Some differences were observed between the European and the Australian samples of L. angustifolia oil and between the two L. x allardii samples. However, whether this is a result of different growing conditions, differences in distillation or lavender cultivar, or age of the oil is unknown. Similar variability in the antibacterial activity of essential oil samples from a given lavender species is reported by Lis-Balchin and Deans (1997) and Lis-Balchin et al. (1998) While these authors suggest differences in their studies may be the result of adulteration of commercial oils to enhance favourable characteristics this is unlikely to be the case in this study as oils were obtained directly from growers. While other studies have investigated the activity of lavandin (L. x intermedia) essential oils the

5 Antibacterial activity of Australian grown Lavandula spp. 13 Table 3 Activity of lavender essential oils against a range of bacteria and the yeast, C. albicans L. angustifolia L. x allardii L. x intermedia L. x heterophylla L. stoechas Avonview Grosso Seal Miss Donnington Double distilled European Australian Single distilled C. freundii 9.1 ± 0.3 No zone 8.2 ± ± ± ± ± ± ± 0.2 E. aerogenes 8.1 ± ± ± ± ± ± ± ± ± 0.3 E. coli 10.0 ± ± ± ± ± ± ± ± ± 0.4 MRSA 11.3 ± ± ± ± ± ± ± ± ± 1.3 P. acnes 13.8 ± ± ± ± ± ± ± ± ± 0.2 Pr. vulgaris 13.5 ± ± ± ± ± ± ± ± ± 2.4 Ps. aeruginosa 6.4 ± 0.9 No zone No zone No zone No zone No zone No zone No zone No zone Sh. sonnei 11.1 ± ± ± ± ± ± ± ± ± 0.7 Staph. aureus 12.9 ± ± ± ± ± ± ± ± ± 0.8 Strep. pyogenes 14.9 ± ± ± ± ± ± ± ± ± 0.7 VRE 10.9 ± ± ± ± ± ± ± ± ± 0.3 C. albicans 9.8 ± ± ± ± ± ± ± ± ± 0.7 Results are presented as mean (±standard deviation) size of zone of inhibition (in mm) including the 6 mm disc. No inhibition was recorded for the water controls. specific cultivar is rarely mentioned (Lis-Balchin and Deans, 1997; Lis-Balchin et al., 1998). In this study we have demonstrated that differences in antibacterial activity exist between different L. x intermedia oils, perhaps due to different chemical composition. In this study the oils of L. x intermedia Miss Donnington and L. x intermedia Seal generally have better antibacterial activity than L. x intermedia Grosso. This may be a consideration when deciding which oil to include in antibacterial products. However, it also be noted that while significant some of these differences are small and whether this reflects a difference in activity in vivo is yet to be determined. No activity was observed with the hydrosol or aqueous extract with Pr. vulgaris the only organism showing significant susceptibility to the ethanolic extracts. Although there have been no other studies published on hydrosols or aqueous extracts of lavender, this data is consistent with work of Shahidi Bonjar et al. (2004) These authors report that methanol extract of leaves from L. stoechas had no effect on the 11 bacteria and 3 yeasts (including E. coli, Ps. aeruginosa, Staph. aureas and C. albicans) included in the assay. We have previously reported that hydrosols and aqueous extracts associated even with very highly antibacterial essential oils have little or no activity (Wilkinson et al., 2003) suggesting that the antibacterial component(s) of essential oils are not water soluble. Some researchers have attempted to correlate antibacterial or antifungal activity with essential oil constituents (Griffin et al., 2000; Lis-Balchin et al., 1998). It has been suggested that antibacterial activity is associated with high concentrations of monoterpenes; however, these authors conclude that the relationship is complex and could not be easily predicted (Lis-Balchin and Deans, 1997; Lis- Balchin et al., 1998). Griffin (2000) in his investigation of the antimicrobial activity of terpenoids showed that antibacterial activity was determined by a complex mix of factors including hydrogen bonding parameters, water solubility and molecular size. In our study no clear relationship was apparent between activity and chemical composition. For example, all hydrosols had high (10 20%) levels of linalool yet did not have any antibacterial activity but in contrast, both L. x allardii and the L. stoechas essential oils had low levels (<5%) linalool and good antibacterial activity. Further work is required to determine the relationship between the various constituents and antibacterial activity. In this study we have demonstrated that Australian grown lavender oils have wide spectrum anti-

6 14 T. Moon et al. bacterial activity in vitro and that no one oil is superior to any other in this respect. As lavender essential is a popular inclusion in toiletry and cleaning products, and has a traditional use as an antibacterial agent, we suggest that further work be undertaken to explore the antibacterial activity of these oils in formulated products in vivo. Acknowledgements The authors thank Bunyip Country Lavender Farm, Crestwood Lavender, Fox Field Lavender, Mt. Wayo Station, Portland Bay Lavender and Dr. Nigel Urwin, CSU Lavender Collection for the donation of lavender plants or products used in this study. This study was funded by a Rural Industries Research and Development Corporation (RIRDC) Grant UCS- 30A. References Catty S. Hydrosols. The next aromatherapy. Vermont: Healing Arts Press; Dadalioğlu I, Everendiliek GA. Chemical composition and antibacterial effects of essential oils of Turkish oregano (Origanum minutiflorum), bay laurel (Laurus nobilis), Spanish lavender (Lavanadula stoechas L.) and fennel (Foeniculum vulgare) on common foodborne pathogens. J Agric Food Chem 2004;52: Field T, Diego M, Hernandez-Reif M, Cisneros W, Feijo L, Vera Y, et al. Lavender fragrance cleansing gel effects on relaxation. Int J Neurosci 2005;115(2): Gattefosse R-M. Gattefosse s aromatherapy. Essex: CW Daniel Company; Griffin S. Aspects of antimicrobial activity of terpenoids and the relationship to their molecular structure. PhD thesis. University of Western Sydney; Griffin S, Markham J, Wyllie SG. Aspects of antimicrobial activity of terpenoids and the relationship to their molecular structure. In: Annual RACI natural products symposium, 6 October 2000, University of New South Wales, Sydney; Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 1999;86(6): Lawless J. The encyclopedia of essential oils. Melbourne, Australia: Element; Lis-Balchin M, Deans SG. Bioactivity of selected plant essential oils against Listeria monocytogenes. J Appl Microbiol 1997;82: Lis-Balchin M, Deans SG, Eaglesham E. Relationship between bioactivity and chemical composition of commercial essential oils. Flavour Frag J 1998;13: Morris N. The effects of lavender (Lavendula angustifolium) baths on psychological well-being: two exploratory randomised control trials. Complement Ther Med 2002;10(4): Peterson L. The Australian lavender industry. A review of oil production and related products. RIRDC Publication No. 02/ 054. Canberra: RIRDC; Shahidi Bonjar GH, Aghighi S, Karimi Nik A. Antibacterial and antifungal survey of plants used in indigenous herbal-medicine of south east regions of Iran. J Biol Sci 2004;4(3): Sosa S, Altinier G, Politi M, Braca A, Morelli I, Della Loggia R. Extracts and constituents of Lavandula multifida with topical ant-inflammatory activity. Phytomedicine 2005;12: Wilkinson JM, Hipwell M, Ryan T, Cavanagh HMA. Bioactivity of Backhousia citriodora: antibacterial and antifungal activity. J Agric Food Chem 2003;51(1):76 81.