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The effect of abiotic growing conditions on antibiosis by extracts of thymus vulgaris.

Introduction

Common thyme, Thymus vulgaris, a member of the mint family. grows both naturally and in cultivation worldwide. In naturally occurring populations the concentration of the major constituents of the essential oil can be linked to biogeographical conditions. Water availability is identified as having the greatest effect on concentration of constituents (Morgan 1989).

DeMartino et al (2009) working in Italy and Pioro-Jabrucka et al (2007) working in New Zealand determined that variant species within the genus Thymus also differ in the relative concentration of the active components in the essential oils. Three of the constituents found in the essential oil: thymol, carvacrol and linalool (Figure 1), have been related to antibiotic activity, with thymol demonstrating the greatest effect ( Cristani 2007, Pioro-Jabrucka 2007).

[FIGURE 1 OMITTED]

Thymol antibiosis has been linked to multiple membrane associated mechanisms of action. In Streptococcus bovis, thymol disrupts membrane integrity, interfering with its transport of glucose (Evans 2000). It alters membrane permeability in Staphylococcus aureus by acting on large unilamellar vesicles composed of phosphatidylcholine/phosphatidylserine (Cristani 2007).

While thymol exposure causes cell death in giardia via plasma membrane alterations which impede osmoregulation, no cytotoxic effects have been observed in mammalian cells (Machado 2010). Thymol acting alone is a less effective antibiotic than thyme constituents acting in combination (Iten 2009, Juven 1994). Thyme essential oils have proven to be more effective against methicillin resistant Staphylococcus aureus (MRSA) and other drug resistant strains than commonly used antibiotics (Warnke 2009).

This study analyses the relative concentrations of thymol in extracts from Thymus vulgaris grown under varying sunlight and water conditions and examines the antibiotic competency of these extracts against Bacillus subtilis, Staphylococcus aureus, MRSA, Escherichia coli, Salmonella spp and Pseudomonas aeruginosa. Control tests were conducted using pure thymol solutions of comparable concentrations to elucidate the role of the thymol component in antibiosis by thyme extracts. Morgan (1989) noted that thyme grown with reduced rainfall and enhanced sunlight produced higher levels of the phenolic compounds thymol and carvacrol, possibly representing the plant's response to water stress.

It is expected that our study will determine that extracts of plants grown in full sun with natural water have greatest antibiosis and highest titers of thymol when compared with extracts from plants grown in shade or in high water conditions. It will further confirm the findings of Iten et al (2009), that the extracts have greater efficacy than equivalent solutions of thymol, establishing that the multi component nature of thyme is essential to its effectiveness as a phytomedicine.

Methods

Thymus vulgaris was grown for three months in outdoor beds consisting of silty clay soil (http:// websoilsurvey.nrcs.usda.gov/app/WebSoilSurvey.aspx) amended before planting with composted horse manure to improve drainage. Experimental beds were placed such that plants received: full sun, full shade, full sun with roadside runoff, all with natural rainfall (see Table 1) and a fourth garden received full sun with daily watering equivalent to 0.5 inches (12.7 mm) rainfall.

All plants were harvested in the autumn after flowering; aerial parts were oven dried at 30[degrees]C overnight and sealed in plastic bags. One gram of herb was extracted in 10.00 mL of 95% ethanol (EtOH) for 72 hours and vacuum filtered through a frittered glass Buchner funnel. The filtrate was concentrated using a Labconco Speedy Vac at 10 mm pressure and 5 x g at 40[degrees]C. Evaporates were reconstituted to 1 mL yielding a final dose equivalent to 1.00 g dried thyme/mL EtOH.

Gas chromatography (GC) analysis was carried out using a GC 2014 Schimadzu Gas Chromatograph with a SHRX5 column having a column length of 15.0 mm and an inner diameter of 0.25 mm to determine the concentration of thymol in extracts of plants from each garden. A 1.0 [micro]L aliquot of 1.00 g herb/mL sample was injected using helium as a carrier gas. The following separation conditions were applied: detector temperature 250[degrees]C, injector temperature 250[degrees]C and initial temperature 100[degrees]C, with a temperature gradient of 5[degrees]C/min, total run time 8 min. Standard curve was performed with 1 [micro]g/mL to 1mg/mL concentrations using a TCD detector.

Antibiosis by the herbal extracts was examined against six bacterial isolates including three gram positive species: Staphylococcus aureus (ATCC 29213), MRSA (patient isolate), and Bacillus subtilis (Wards 85V0228) and three gram negative species: Escherichia coli (ATCC 25922), Salmonella spp (patient isolate) and Pseudomonas aeruginosa (ATCC 27853). Isolates were inoculated in 30 mL of nutrient broth in 250 mL nephalo flasks and incubated at 150 rpm and 37[degrees]C for 24 hours. Cell population was spectrophotometrically determined for each culture and bacterial stocks were diluted to produce McFarland 0.5 standards for plate inoculation. Bacterial lawns were applied to Mueller-Hinton agar.

Kirby Bauer antibiotic sensitivity testing was performed using 6 mm disks composed of Whatman #3 filter paper impregnated with 100 [micro]L, 50 [micro]L, 20 [micro]L, 10 [micro]L, 5 [micro]L or 2 [micro]L of thyme extract. Disks impregnated with 100 [micro]L 95% ethanol served as controls. Plates were incubated for 24 hours in 5% C[O.sub.2] at 37[degrees]C prior to measuring zones of inhibition. Corresponding minimum inhibitory concentrations (MICs) were ascertained by culturing the S. aureus, MRSA and B. subtilis in Mueller-Hinton broth and preparing a standardised inoculum. Thyme extracts were serially diluted 1:2 such that addition of the standard inoculum resulted in 5.0 x [10.sup.5] cells and 5.0 x [10.sup.-1] to 3.12 x [10.sup.-4] mL extract per mL broth. All microbes were incubated at 37[degrees]C in 5% C[O.sub.2] for 18 hours. The last tube in a dilution series not demonstrating growth was recorded as the MIC. Aliquots (10 [micro]L) from inhibitory dilutions were applied to trypticase soy agar (TSA) with 5% sheep red blood cells (SRBC) and incubated 24 hours in 5% C[O.sub.2] at 37[degrees]C to determine minimum bactericidal concentrations (MBCs).

Two approaches were taken to determine the relative effectiveness of thyme extract to pure thymol solution. Thymol solutions were made in concentrations to correspond to the amount of thymol found in each extract (0.659 mg/mL for sun extract, 0.429 mg/mL for roadside, 0.320 mg/mL for shade and 0.205 mg/mL in extracts from plants watered daily). Paper discs were prepared with 100 [micro]L, 50 [micro]L, 20 [micro]L, 10 [micro]L, 5 [micro]L or 2 [micro]L of thymol solutions for Kirby Bauer analysis using all three gram positive bacterial species. The diameters of the zones of inhibition were compared with equivalent thyme extract results to determine the thymol specific effect in antibiosis versus its potentially synergistic role with the other constituents of the essential oil.

In a second approach, solutions were prepared at equivalent concentrations as stated above and in concentrations equal to 5, 10 or 50 times the concentration of thymol found in the extract. A quantity of 100 [micro]L of each thymol solution, 100 [micro]L of thyme extract and 100 [micro]L of ethanol alone were applied to 6 mm paper disks for Kirby Bauer analysis using all six bacterial species. MIC and MBC analyses of the thymol dilutions were also performed. Significance of differences among treatments was tested by applying one way ANOVA. When the ANOVA null hypothesis was rejected, post hoc comparisons were performed by Students 1 tailed t-tests.

Results

The retention time for thymol in the GC column was 3.76 minutes. Greatest amounts of thymol were found in full sun/natural water (0.659+/-0.17 mg thymol/mL extract), followed by extracts from roadside plants/ natural water (0.429+/-0.08 mg/mL), shade/natural water plants (0.320+/-0.06 mg/mL extract) and full sun/wet conditions (0.204+/-0.03 mg/mL extract) (see Figure 2). Thymol concentrations of plants subjected to daily watering were significantly lower than concentrations from those grown under natural water conditions, by t-tests p<.05.

Ethanol extracts from each of the four growing conditions using 100 [micro]L of extract per disk showed significantly greater antibiosis in full sun, full shade and roadside gardens grown with natural water than those grown in full sun but watered daily (ANOVA p<0.01 with the garden watered daily significantly different from each other garden by t-test p<.05). However no significant differences were seen between any of the natural water gardens despite the obvious trend of greatest antibiosis in extracts from full sun followed by full sun roadside and trailed by shade garden plants (see Figure 3).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

For all growing conditions, when disks of differing volumes of extract (increasing from 2 [micro]L extract per disk to 5 [micro]L, 10 [micro]L, 20 [micro]L, 50 [micro]L and 100 [micro]L) were tested using a control of EtOH only, the zone of inhibition increased in direct proportionality to the dose (see Figure 4).

For the plants grown in natural water conditions starting with control vs 2 [micro]L, each dose was significantly different from the next by Student's 1 tailed t-test (p<0.05). Plants watered daily showed no significant difference from control to the 2 [micro]L, 5 [micro]L or 10 [micro]L dose, but the 20 [micro]L dose was significantly different from the 10 [micro]L, and all subsequent doses were significantly different from the next in the series by Student's 1 tailed t-test p<0.05). Significant difference in antibiosis was achieved by the three sets of plants grown with natural water conditions and those watered daily (p<0.05) but no statistical significance between plants grown in shade, full sun or roadside conditions (See Figure 5) although the aforementioned trend was noted.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Antibiosis by thyme extracts was seen in all gram positive species. Although the differences were not significant, a distinct trend was observed with extracts inhibiting growth most effectively in MRSA, slightly less so in S. aureus and less again in B. subtilis. Extracts inhibited growth minimally in E. coli and not at all in either Salmonella spp or P. aeruginosa. MIC values were highest against MRSA (0.025 g/mL in both road and sun plants with corresponding MBC values of 0.05 g/mL) but were not seen for the extracts from shade or sun with daily water gardens (N=3). Testing of thymol alone, in concentrations equivalent to those in each of our extracts, showed a dose dependent tenfold decrease in antibiosis when compared to the extract itself, accompanied by a tenfold decrease in concomitant MIC/ MBC values (See Table 2).

[FIGURE 6 OMITTED]

Kirby Bauer testing revealed that thyme extracts were more effective than thymol alone in inhibiting growth of gram positive species (see Figure 6 A, B, C), approximately equivalent to thymol alone against E. coli (see Figure 6 D), less effective than thymol alone against Salmonella (see Figure 6 E) and neither the thymol (even at doses fifty times that found in the herb) nor the thyme itself exhibited antibiosis against P. aeruginosa (see Figure 6 F). Zones of inhibition surrounding disks containing 100 [micro]L of thymol solution at the equivalent dose to that found in the herb (1x) or five, ten or fifty times greater dose (5x, 10x or 50x) were plotted. The zone of inhibition associated with 100 [micro]L of thyme extract was compared with this standard curve to determine the relative effectiveness of thyme extract compared with known thymol doses (see Figure 7).

[FIGURE 7 OMITTED]

Thymol had its greatest efficacy against MRSA where zones of inhibition were graphically extrapolated to represent a mean of twenty five times the effectiveness of thymol alone (n=4) compared with extract being sixteen times more effective than the equivalent dose of thymol against S. aureus (n=4) (see Figure 8). We have consistently seen a lower efficacy of thyme extract against the spore forming bacterium B. subtilis, but even here it is seven times as effective as thymol alone for levels of extract seen in plants from full sun natural water conditions. However thymol alone is ineffective at the lower doses seen in plants grown under other garden conditions.

[FIGURE 8 OMITTED]

Discussion

The very obvious differences in thymol content of extracts from plants grown under differing abiotic conditions were to be expected and were comparable to those noted by Morgan et al (1989). A probable explanation for the most significant difference is water stress. This is in keeping with the findings of Boscaiu et al (2009) who observed increases in osmolytes such as proline, sugars and quaternary ammonium compounds, in plants grown under arid conditions. Therefore it seems plausible that our plants grown with reduced water availability would result in the elevated levels of phenols demonstrated by thymol quantification.

The levels of thymol which exhibited antibiosis against S. aureus were consistent with those found by Juven et al (1994). This study determined that thyme extract was sixteen times more effective than thymol alone in inhibiting growth of S. aureus. Warnke (2009) found thyme extracts to be one of the most effective herbal antibiotics against MRSA. Our study found thyme extracts to be twenty five times more effective against MRSA than equivalent concentrations of thymol in solution. These findings lend credence to the argument of Iten (2009) that there is synergistic action of the constituents of herbal extracts which render the herb as a whole to be a more effective antibiotic than its individual components.

Thyme essential oil has a combination of constituents including thymol, carvacrol, linalool and cymene (which has no independent antibiotic activity) (DeMartino, 2009, Morgan 1989, Pioro-Jabrucka 2007) that most likely working synergistically and exhibited dose dependent antibiosis against the gram positive but not the gram negative species examined. Structural features of the outer membrane of gram negative bacterial cell walls may be preventing the thymol from crossing, which would explain the reduced antibiosis when we tested E. coli, and absence of antibiosis against Salmonella and P. aeruginosa. Although others have reported antibiosis against gram negative bacteria, their findings, like ours, determined thyme to be consistently more effective against gram positive species (Pioro-Jabrucka 2007); as is thymol which acts on the plasma membranes of gram positive bacteria and giardia (Cristani 2007, Evans 2000, Machado 2010).

Cristani et al (2007) observed an almost detergent like action of thymol against the phosphatidylcholine component predominant in the plasma membrane of gram positive bacterial species. If the mechanism of thymol antibiotic activity requires interaction with phosphatidylcholine, the reduced concentration of this phospholipid in the outer membrane of the gram negative wall could again explain the ineffectiveness of thyme extracts at inhibiting the growth of these microbes. Alternately Evans et al (2000) suggest that thymol disrupts membrane integrity by altering protein reactions in gram positive species, possibly acting as ionophores. Machado et al (2010) working with giardia, postulated that thymol altered plasma membrane osmoregulation without impacting mammalian plasma membrane function. Thus thymol is likely acting to disrupt membrane permeability but the mechanism is yet to be elucidated.

Significantly greater antibiosis was noted by extracts of plants watered only by rainfall than by extracts of plants watered daily. The decreases in antibiosis by plants grown in natural water but full shade conditions may also be attributed to moisture content as gardens in shade retained water longer than gardens in full sun. There are additional conditions which may be contributory, including levels of other thyme components and abiotic factors such as nutrient content of the soil. We observed good correlation of dose dependent antibiosis by thymol as a purified standard and a tenfold greater dose dependent antibiosis by thyme extracts. It can be presumed that the increased efficacy of the extracts is due to additive effects of other active components.

Conclusion

Thyme is a good antibiotic. Whole plant extract is more effective than thymol working alone. Thyme has potential to be therapeutically developed against multiply drug resistant microbial species. When pursuing this development, conditions under which the thyme is grown will be critical to producing plants with greatest antibiotic potential.

Acknowledgements

The authors would like to thank Drs Victor W Motz and Tevye Celius of the Department of Chemistry at Ohio Northern University and Rachel Yanikov for their invaluable assistance in this endeavour; and United Plant Savers for their support of herbal education and research at ONU.

References

Boscaiu M, Mora E, Fola O, Scridon S, Llinares J, Vicente O. 2009. Osmolyte accumulation in xerophytes as a response to environmental stress. UASVM Horticulture 6:1;96-102.

Cristani M, D'Arrigo M, Mandalari G, Micieli D, Venuti V, Bisignano G, Saija A, Trombetta D. 2007. Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity. J Agric Food Chem 15;6300-8. EPub 30 June 2007.

DeMartino L, Bruno M, Formisano C, DeFeo V, Napolitano F, Rosselli S, Senatore F. 2009. Chemical composition and antimicrobial activity of essential oils from two species of Thymus growing in southern Italy. Molecules 14:11;4614-24.

Evans JD, Martin SA. 2000. Effects of thymol on ruminol microorganisms. Curr Microbiol 41;336-40.

Iten F, Saller R, Abel G, Reichling. 2009. Additive Microbial effects of the active components of the essential oil of Thymus vulgaris--chemotype carvacrol. Planta Med 11;1231-6. EPub 3 April 2009.

Juven BJ, Kanner J, Schved F, Weiss-Lowicz H. 1994. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J ApplMicro 76:6;626-31.

Machado M, Dinis AM, Salgueiro L, Cavaleiro C, Custodio JB, Sousa MD. 2010. Anti-giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris and Lippa graveolens on trophozoites growth, viability, adherence and ultrastructure. ParisitolRes EPub March 2010.

Morgan RK. 1989. Chemotypic characteristics of Thymus vulgaris L. in Central Otago New Zealand. J Biogeography 16:5;483-91.

Pioro-Jabrucka E, Suchorska-Tropilo K, Rzewuska M. 2007. Antibacterial activity of the essential oil of common thyme Thymuspulegiodes L. and wild thyme Thymus serpyllum L. Herba Polonica 53:3;297-301.

Warnke PH, Becker ST, Podschun R, Sivanantnan S, Springer IN, Russo PA et al. 2009. The battle against multi-resistant strains: renaissance of antimicrobial essential oils as a promising force to fight hospital acquired infections. J Craniomaxillofac Surg 37:7;392-7. EPub 26 May 2010.

* Vicki Abrams Motz (1), Linda M Young (1), David H Kinder (2)

(1) Department of Biological and Allied Health Sciences, Ohio Northern University, 525 S Main St, Ada OH 45810 USA

(2) Raabe College of Pharmacy, Ohio Northern University, 525 S Main St, Ada, OH 45810 USA

* Corresponding author: v-motz@onu.edu, phone 1 491 772 2063, fax 1 419 772 2330
Table 1: High temperature and precipitation for
1 June to 31 August 2009 at the closest weather
station to the longitude and latitude of the
experimental gardens

(http://www.farmersalmanac.com/weather-history/43040)

                                  [degrees]F/   [degrees]C/
                                    inches           mm

Mean high temperature             83.1 + 5.1    28.4 + (-14)
Mean daily rainfall               0.13 + 0.33    3.3 + 8.4
Number of days with no rainfall              57

Table 2: Comparison of MIC/MBC values (g/mL) for antibiosis by
thyme extracts (n=3) and solutions of equivalent amounts of
thymol alone from plants grown under differing conditions against
three gram positive strains

                Sun          Road          Shade        Wet
        Sun    equiv  Road   equiv  Shade  equiv  Wet  equiv

Methicillin resistant Staphylococcus aureus
 MIC   0.025    0.5   0.025  0.25    NA    0.25   NA    0.5
 MBC    0.05    0.5   0.05    0.5    NA     NA    NA    NA

Staphylococcus aureus
 MIC    0.05    0.5   0.025  0.25    NA    0.25   NA    0.5
 MBC    0.05    0.5   0.05    0.5    NA     NA    NA    NA

Bacillus subtilis
 MIC   0.025   0.25   0.05    0.5    NA     0.5   NA    NA
 MBC   NA (1)  0.25   0.05    0.5    NA     0.5   NA    NA

(1) NA = no antibiosis seen at highest dose
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Title Annotation:Growing and manufacturing
Author:Motz, Vicki Abrams; Young, Linda M.; Kinder, David H.
Publication:Australian Journal of Medical Herbalism
Article Type:Report
Geographic Code:1USA
Date:Dec 22, 2010
Words:3359
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