Fitoterapia 75 (2004) 192–200
Antimicrobial activity of Artemisia douglasiana leaf essential oil William N. Setzera,*, Bernhard Voglera, Jennifer M. Schmidta, Joseph G. Leahyb, Richard Rivesc a Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA c Wyatt Archaeological Research, Cornersville, TN 37047, USA
b
Received 3 June 2003; accepted 18 December 2003
Abstract Artemisia douglasiana leaf has been shown to be efficacious complementary herbal treatment for chronic bladder infection in a paraplegic youth. The leaf oil has been analyzed by GC-MS and the major components found to be camphor (29%), artemisia ketone (26%), artemisia alcohol (13%), a-thujone (10%), 1,8-cineole (8%), and hexanal (5%). The leaf oil and the major components have been tested for antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The essential oil shows limited antimicrobial activity in vitro, so it is unclear if the oil exerts a direct antimicrobial effect in vivo, or plays some role in stimulation of host defenses. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Artemisia douglasiana; Essential oil; Antimicrobial; Bladder infection
1. Introduction Many members of the genus Artemisia (Asteraceae) are important medicinal plants. Thus, for example, A. vulgaris (mugwort), native to Britain and Europe, has been used as a tonic, febrifuge, anthelmintic, female troubles, nervous disorders, *Corresponding author. Tel.: q1-256-824-6519; fax: q1-256-824-6349. E-mail address:
[email protected] (W.N. Setzer). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2003.12.019
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complaints of the gastrointestinal tract (e.g. stomach ulcers and indigestion) w1x. The essential oil shows antimicrobial activity and contains 1,8-cineole, camphor, and thujone as major constituents w1x. Similarly, A. absinthium (wormwood) w2x has been used in Europe and A. mexicana w3x in Mexico to stimulate the appetite and aid digestion. Artemisia douglasiana Besser (California mugwort) is a common perennial herb found throughout the Western United States, especially northern California, Oregon, and Washington, 1–800 m elevation, and prefers dry, partially shaded streamsides or riverbanks on most geologic formations. A. douglasiana and A. vulgaris have similar medicinal uses; A. douglasiana is used to promote menstruation, as a stimulant, tonic, to treat nervous disorders, and as a diuretic w1x. The essential oil has been used for aromatherapy, inhaled for mental clarity and ease of mental distress; used as a massage for aching muscles and pain on the surface of the body; and as a bath or tonic. Recurrent urinary tract infections are generally treated with antibiotics. b-Lactams such as amoxicillin or bacampicillin effectively clear up acute bladder infections, but can adversely affect normal urogenital flora w4x. Fluoroquinolone antibiotics such as norfloxacin w5x or ciprofloxacin w6x have been used for prophylactic treatment of recurrent urinary tract infections. In this paper, we describe the use of A. douglasiana leaf for the prophylactic treatment of chronic bladder infections in a paraplegic youth, and we present the chemical analysis and antimicrobial activity of A. douglasiana leaf essential oil. 2. Case report An adolescent white male with myelomeningocele spina bifida has had a recurrent urinary tract infection since 1998 resulting from the introduction of P. aeruginosa (identified by enterotube analysis at the Microbiology Department, University of North Carolina at Charlotte) into the urinary tract during a urodynamic test. Previous and ongoing prescribed antibiotic treatments include ceftazadime injections and oral ciprofloxicin, but infection recurs after a few days. To augment the prescribed antibiotic treatment, whole A. douglasiana leaves have been administered (one tablespoon leaves, chewed for 10 min and then swallowed, three times per day). After 2–5 days of A. douglasiana herbal treatment, symptoms of infection generally subside. Microscopic evaluation of the urine after A. douglasiana treatment showed that the P. aeruginosa was still present in the urine, but the bacterium grew in chains rather than single or double individuals (Fig. 1), suggesting the possibility that the treatment initially caused some sublethal effect on cell division and subsequent cell detachment. To our knowledge, this growth form of P. aeruginosa has not been previously reported in the literature. After a period of approximately 2 weeks, the presence of virulent non-chain bacteria in the urine become negligible and all visible indications of urinary tract infection subside. After 3 or 4 months, however, immediate secondary infection attributed to Citrobacter freundii occurs requiring immediate antibiotic treatment.
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Fig. 1. Pseudomonas aeruginosa, chain-growth morphology.
3. Experimental 3.1. Leaf essential oil A. douglasiana leaves were obtained from Dave Keoppel, Santa Barbara, California. The essential oil was obtained by hydrodistillation of the chopped leaves (59.05 g) followed by dichloromethane extraction of the distillate to afford 204.8 mg leaf oil. 3.2. Gas chromatographic-mass spectral analysis The essential oil of A. douglasiana was subjected to GC-MS analysis on an Agilent system consisting of a model 6890 GC, a model 5973 mass selective detector, and an Agilent ChemStation data system. The GC column was an HP-5 ms fused silica capillary with a (5% phenyl)-methylpolysiloxane stationary phase, film thickness of 0.25 mm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mlymin. Inlet temperature was 200 8C and MSD detector temperature was 280 8C. The GC oven temperature program was used as follows: 40 8C initial temperature, hold for 10 min; increased at 38ymin to 200 8C; increased 28ymin to 220 8C. The sample was dissolved in CH2Cl2 and a split injection technique was
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Table 1 GC-MS analysis of A. douglasiana leaf essential oil Compound
Confidence level (%, NIST database)
Area %
RI (this work)
RI Ref. w22x
Hexanala Camphenea Yomogi alcohol 1,8-Cineolea Artemisia ketonea Artemisia alcohola a-Thujonea b-Thujonea Camphora endo-Borneola Terpinen-4-ola Myrtenala
94 97 91 98 78 64 97 97 97 91 95 91
5.27 1.03 3.09 7.53 26.09 13.15 9.68 0.93 28.98 2.07 1.14 1.03
808 952 1006 1031 1070 1089 1101 1116 1147 1165 1177 1194
800 953 998 1033 1062 1083 1102 1114 1143 1165 1177 1193
a
Verified by co-injection of authentic compound.
used. The components from the gas chromatographic-mass spectral analysis are reported in Table 1. 3.3. Antimicrobial activity The essential oil and the individual components were screened for antimicrobial activity against Gram(q) bacteria, B. cereus (ATCC No. 14579) and S. aureus (ATCC No. 29213); Gram (y) bacteria, P. aeruginosa (ATCC No. 27853) and E. coli (ATCC No. 25922); and the fungi C. albicans (ATCC No. 10231) and A. niger (ATCC No. 16401). Minimum inhibitory concentrations (MIC) were determined using the microbroth dilution technique w7,8x. The antimicrobial activities of the essential oil and components are reported in Table 2. In order to further probe the antibacterial effects of the essential oil and its major components, we have determined the growth rates at sublethal concentrations for B. cereus and P. aeruginosa. Growth curves were carried out in triplicate using 96well microplates. Dilutions of A. douglasiana essential oil and the major components were prepared in cation-adjusted Mueller Hinton broth (CAMHB) beginning with 50 ml of 1% wyw solutions of compounds in DMSO plus 50 ml CAMHB. The extract solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5=108 colony forming units (CFU)yml were added to each well. Plates were incubated at 37 8C and turbidity (by measuring optical density at 600 nm) recorded every 20 min for 8 h. The final MIC was determined as the lowest concentration without turbidity. Growth data (log OD vs. time) were plotted, the slopes of the exponential growth phase determined, and average slopes and standard deviations calculated based on the three replicates. Statistical analysis of the data was done using the SYSTAT Version 10 program (Richmond, CA, USA).
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Table 2 Antimicrobial activities (MIC, mgyml) of A. douglasiana leaf essential oil and components Compound
Bacillus cereus
A. douglasiana essential oil Artemisia alcohol Artemisia ketone Endo-Borneol Camphor 1,8-Cineole Hexanal Myrtenal 4-Terpineol a-Thujone b-Thujone Positive control
156
a b
312 625 312 312 312 78 156 312 312 312 1.22a
Staphylococcus aureus 625 1250 1250 625 1250 1250 625 625 1250 1250 1250 0.61a
Escherichia coli
Pseudomonas aeruginosa
Candida albicans
625
625
625
625 625 625 625 625 625 625 625 625 625 1.22a
625 625 625 625 625 625 625 625 625 625 2.44a
625 625 625 625 625 625 312 312 625 625 0.61b
Aspergillus niger 625 625 1250 625 625 625 2500 1250 625 1250 1250 1.22b
Gentamicin sulfate. Amphotericin B.
We also have carried out additional in vitro experiments to determine whether A. douglasiana essential oil or its major components offer protection to bladder cells against P. aeruginosa infection. Thus, 5637 (human primary bladder carcinoma) cells were plated into 96-well cell culture plates at 5.1=103 cells per well w8x. After 48 h, supernatant fluid was removed by suction and replaced with 100 ml growth medium containing 1.0 ml of solutions of A. douglasiana essential oil or compounds in DMSO (1.0%), yielding final concentrations of 100 mgyml for the compounds. The DMSO solutions of compounds were added to wells in four replicates. Medium controls and DMSO controls (25 ml DMSOyml) were used. P. aeruginosa bacteria at a concentration of approximately 1.5=108 colony forming units (CFU)yml were added to each well. The wells were microscopically inspected after 24 h. 4. Results and discussion Various Artemisia essential oils as well as the major components found in A. douglasiana leaf essential oil have previously shown antimicrobial activity. Thus, for example, A. caerulescens w9x, A. mexicana w10x, A. afra w11x, and A. asiatica w12x have been reported to exhibit antibacterial activity, and many are used as herbal medicines. Moreover, the most abundant component in the leaf essential oil of A. douglasiana, camphor, has been reported to exhibit bacteriostatic activity against P. aeruginosa w13x, and this compound in a major constituent in a number of antibacterial essential oils w14,15x. 1,8-Cineole w16,17x, 4-terpineol w18–20x, and endo-borneol w21x have been previously reported to exhibit antibacterial and antifungal activity. In the present study, the results of experiments conducted using B. cereus (Table 3) indicate that while neither the essential oil of A. douglasiana nor its individual
Conc (mgyml)
Control
Ess oil
Artemisia alcohol
Artemisia ketone
Borneol
Camphor
Cineole
Hexanal
Myrtenal
Terpinen-4-ol
a-Thujone
b-Thujone
2500 1250 625 312 156 78 39 20
* * MIC 0.205 0.255 0.503 0.551 0.557
* * * * MIC 0.358 (0.027) 0.521 (0.008) 0.521 (0.113)
* * * MIC 0.225 0.460 0.443 0.512
* * MIC 0.256 0.381 0.437 0.540 0.553
* * * MIC 0.197 0.385 0.393 0.469
* * * MIC 0.262 0.388 0.389 0.428
* * * MIC 0.290 0.377 0.375 0.437
* * * * * MIC 0.356 (0.022) 0.374 (0.010)
* * * * MIC 0.326 (0.007) 0.326 (0.065) 0.338 (0.013)
* * * MIC 0.314 0.351 0.467 0.479
* * * MIC 0.371 0.407 0.434 0.438
* * * MIC 0.334 0.417 0.497 0.519
(0.007) (0.002) (0.006) (0.052) (0.046)
(0.005) (0.033) (0.040) (0.015)
(0.001) (0.026) (0.022) (0.042) (0.020)
(0.011) (0.021) (0.008) (0.056)
(0.003) (0.019) (0.017) (0.011)
(0.003) (0.033) (0.043) (0.026)
Standard deviations are shown in parentheses. Data in bold represent means judged to be significantly different from the control mean (as0.05).
a
(0.034) (0.041) (0.031) (0.025)
(0.018) (0.022) (0.043) (0.066)
(0.026) (0.013) (0.025) (0.023)
W.N. Setzer et al. / Fitoterapia 75 (2004) 192–200
Table 3 Exponential growth rates for B. cereus with A. douglasiana essential oil and componentsa
197
198
Conc (mgyml)
Control
Ess Oil
Artemisia alcohol
Artemisia ketone
Borneol
Camphor
Cineole
Hexanal
Myrtenal
Terpinen-4-ol
a-Thujone
b-Thujone
2500 1250 625 312 156 78 39 20
* * MIC 0.244 0.244 0.278 0.284 0.277
* * MIC 0.248 0.213 0.253 0.278 0.278
* * MIC 0.261 0.231 0.264 0.289 0.271
* * MIC 0.184 0.257 0.281 0.287 0.269
* * MIC 0.168 0.203 0.278 0.271 0.257
* * MIC 0.182 0.233 0.277 0.258 0.254
* * MIC 0.192 0.228 0.270 0.277 0.247
* * MIC 0.252 0.212 0.238 0.270 0.244
* * MIC 0.230 0.291 0.310 0.283 0.271
* * MIC 0.223 0.260 0.293 0.277 0.265
* * MIC 0.217 0.275 0.330 0.294 0.266
* * MIC 0.222 0.267 0.321 0.299 0.263
(0.011) (0.011) (0.013) (0.003) (0.010)
(0.018) (0.008) (0.007) (0.003) (0.009)
(0.012) (0.014) (0.011) (0.010) (0.007)
(0.005) (0.006) (0.004) (0.011) (0.007)
(0.013) (0.007) (0.004) (0.022) (0.008)
(0.022) (0.009) (0.006) (0.008) (0.012)
(0.017) (0.006) (0.008) (0.009) (0.011)
(0.015) (0.009) (0.020) (0.022) (0.005)
Standard deviations are shown in parentheses. Data in bold represent means judged to be significantly different from the control mean (as0.05).
a
(0.006) (0.012) (0.011) (0.005) (0.014)
(0.010) (0.004) (0.006) (0.006) (0.007)
(0.005) (0.011) (0.007) (0.014) (0.005)
(0.021) (0.015) (0.006) (0.018) (0.010)
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Table 4 Exponential growth rates for P. aeruginosa with A. douglasiana essential oil and componentsa
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components exhibited appreciable antimicrobial activity as judged by MIC determinations, the aforementioned compounds and other components inhibited growth rates in this organism. Borneol, camphor, 1,8-cineole, hexanal, myrtenal, terpinen4-ol, and a-thujone all decreased growth rates significantly (as0.05) at 20 mgyml. P. aeruginosa appeared to exhibit greater resistance to these effects (Table 4), although hexanal, 1,8-cineole, and camphor inhibited growth rates at some concentrations. Visual inspection revealed that in vitro infection of human bladder tumor cells with P. aeruginosa caused complete disruption of cellular growth and morphology of the bladder cells, indicating that neither A. douglasiana essential oil nor the major components studied provided any protection against P. aeruginosa under the described conditions. 5. Conclusions Artemisia douglasiana leaf has been shown, in this particular case, to be an effective complementary treatment for recurrent urinary tract infection. Components of the leaf oil exhibited relatively high MIC’s against B. cereus and P. aeruginosa, but to varying degrees, significantly inhibit growth rates in both strains. Neither A. douglasiana leaf oil nor its components showed in vitro protection of bladder cells against P. aeruginosa infection. Based on these findings, we cannot state with certainty whether the efficacy of A. douglasiana herbal treatment for chronic bladder infection is or is not due to some bacteriostatic activity of the essential oil or its components. The relatively weak antimicrobial activity observed in in vitro analyses may not reflect the effects of the oil and its components in vivo, considering, for example, synergistic effects with host compounds or structures. Alternatively, however, the efficacy of the oil in treatment of bladder infection may be the result of antimicrobial activity by non-volatile components of the leaf, or other effects, such as stimulation of host defenses. Further research is necessary in order to determine the mechanism of the herb in inhibiting P. aeruginosa infection and the components responsible for the bioactivity. Acknowledgments This work was made possible by a generous grant from an anonymous private donor. References w1x Gruenwald J. PDR for herbal medicines. 2nd ed. Montvale, NJ: Thompson Medical Economics Company, 2000. w2x Bruneton J. Pharmacognosy, phytochemistry, medicinal plants. 2nd ed. London: Intercept Ltd, 1999. w3x Morton JF. Atlas of medicinal plants of Middle America. Springfield, IL: Thomas Publishers, 1981. w4x Reid G, Bruce AW, Cook RL, Llano M. Scand J Infect Dis 1990;22:43.
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