African Journal of Biotechnology Vol. 8 (7), pp. 1280-1286, 6 April, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals

Full Length Research Paper

The proposed mechanism of bactericidal action of eugenol,
-terpineol and γ-terpinene against Listeria monocytogenes, Streptococcus pyogenes, Proteus vulgaris and Escherichia coli

S. O. Oyedemi*, A. I. Okoh, L. V. Mabinya, G. Pirochenva and A. J. Afolayan

School of Biological Sciences, University of Fort Hare, Alice 5700, South Africa.

Accepted 19 November, 2008

The mechanism of antimicrobial activity of essential oils components;
- terpineol, γ-terpinene and eugenol was studied to evaluate their effect on the bacterial membrane against four strains of bacteria: Listeria monocytogenes, Streptococcus pyogenes, Proteus vulgaris and Escherichia coli. The study was done to observe changes in membrane composition by assaying for the leakage of protein and lipid using Bradford and van Handel’s method respectively. The oils components were capable of inducing cell lysis by the leakage of protein and lipid contents. Eugenol at 2 × MIC was highly effective toward protein content leakage after 120 min of exposure. Alpha terpineol and γ-terpinene showed similar effect at 2 × MIC under the same condition. Gamma terpinene displayed the highest activity toward lipid content leakage at 2 x MIC while
-terpineol and eugenol showed similar effect after 120 min of exposure. The result revealed that both cell wall and membrane of the treated gram negative and gram positive bacteria were significantly damaged.

Key word: Mechanism of action, essential oil components, lipid content, protein content.

INTRODUCTION

The probable mechanism of action of antibacterial activity have been made to elucidate this mechanism in Gram of essential oil previously studied was investigated. The negative and Gram positive bacteria (Ultee et al., 1998). antibacterial activity of essential oils studied was Furthermore, little or no work has been done on the supported by the results obtained by gas-liquid chromato- mechanism of actions of
- terpineol, γ-terpinene and graphic analysis. The chemical analysis revealed the eugenol on the protein and lipid leakage of bacterial presence of eugenol in Ocimum basilicum and Pteronia membrane. incana oils,
-terpineol and γ-terpinene was present in The aim of this study is to investigate the role of Rosemary officinallis oil, while
-terpineol was present in eugenol, γ-terpinene and
–terpineol played in the inhibi- Eucalyptus cinerea (yet to be published).
-terpineol, γ- tion of Listeria monocytogenes, Streptococcus pyogenes, terpinene and eugenol were present as major compo- Proteus vulgaris and Escherichia coli at bactericidal nents of the used essential oils. The presence of these concentration. Likewise, to evaluate the mechanism of components may constitute the effectiveness of the oils inhibition by studying their potential of inducing cell lysis based on their structural configuration (γ-terpinene and through protein and lipid leakage. eugenol) and their relative percentage composition (Marino et al., 2001). However, the basis of the mecha- MATERIALS AND METHODS nisms of action of essential oils and their components has not been fully established. Recent investigations Bacteria strains used in this study

The reference strains used in this study were chosen based on their pathological effects on human and deterioration of food products: *Corresponding author. E-mail: [email protected]. Gram positive bacteria; Listeria monocytogens (ATCC 12022) and Oyedemi et al. 1281

Table 1. Bactericidal activity of essential oil components against selective bacteria.

Nature of inhibition (%)

γ-terpinene
-terpineol Eugenol

Log kill Log kill Log kill Log kill Log kill Log kill Test organisms MIC MIC MIC 1×MIC 2×MIC 1×MIC 2×MIC 1×MIC 2×MIC L. monocytogenes 0.50 3.175§ 7.79§ 0.50 1.49 2.40 0.50 3.23§ 7.84§ S. pyogenes 0.50 8.30§ 7.60§ 0.25 1.50 2.79 0.25 3.26§ 3.60§ P. vulgaris 0.75 1.48 2.21 0.50 2.94 4.07§ 0.50 2.84 7.90§ E. coli 0.50 2.50 3.26§ 0.75 4.70§ 7.00§ 0.25 2.22 7.00§

MIC represents Minimum inhibitory concentrations; § represents bactericidal activity.

Streptococcus pyogenes (ATCC 19615). Gram negative bacteria; 30 min intervals each suspension was centrifuged at 7000 rpm. Escherichia coli (ATCC 87536) and Proteus vulgaris (ATCC 43071) Protein amounts were determined at 595 nm using Coomassie were obtained from the Institute of Louis Pasteur, Paris, France. brilliant blue G-250 by the method of Bradford (1976). The concen- tration of protein leakage was extrapolated from Bovine serum albumin (BSA) which was used as a standard. Essential oil components

Eugenol, γ-terpinene and
-terpineol of essential oil components Lipid leakage assay used were purchased from Sigma-Aldrich Steinheim (Germany). They were used based on the result of GC-MS chromatography Lipid leakage was measured using a method described by van obtained from previous study. Handel (1985). Bacterial cultures were harvested after standardi- zation (1.2 × 108 CFU/ml) by centrifugation at 10000 rpm. The cell suspension treated with 1 × MIC and 2 × MIC concentrations of Culture media and growth conditions eugenol,
-terpineol and γ-terpinene was further incubated at 37oC for 30 min. At each time interval each suspension was centrifuged The bacterial stock cultures were maintained on nutrient agar at 10000 rpm for 10 min. The absorbance of the duplicate samples (Saarchem, Gauteng, SA) plates. A loopful of bacterial cells from after the addition of vanillin-phosphoric acid reagent followed by the nutrient agar plates was inoculated into 100 ml nutrient broth vortexing was measured at 525 nm after 30 min. The concentration (Difco, California, USA) in 250 ml side arm Erlenmeyer flask and o of lipid leakage was estimated from the triolein standard curve. incubated at 37 C for 16 h with vigorous shaking (orbital incubator, S150, UK). After incubation, the culture was diluted with fresh media to give an O.D 600nm of 0.1. One hundred micro litre of the culture cells was added onto the plate and spread into a bacterial RESULTS lawn using a sterile glass spreader. Antibacterial and time-kill regimes of eugenol, γ- terpinene and
-terpineol MIC determination of eugenol, γ-terpinene and
-terpineol

The Minimum Inhibitory Concentration (MIC) of the essential oils The result of the time–kill studies is summarized in Table and its components was determined by broth dilution technique as 1. The data are presented in terms of the log-CFU/mL described by Irobi et al. (1996). Essential oil components were and are judged relative to the convectional definition of diluted with Tween-80 to give concentrations ranging from 0.01 to bactericidal activity, that is, 3-log-CFU/mL or greater 5 2% v/v. 50 l of standardized 18 h incubated bacterial culture (10 reduction in the initial inoculum within 24 h (Yaki and CFU/ml) was introduced into test tubes, followed by the addition of essential oils components; eugenol,
-terpineol and γ-terpinene. A Zurenko, 2003). At 2 × MIC, γ-terpinene demonstrated set of tubes containing only growth medium inoculated with each of bactericidal activity against all the strains tested except P. the bacterial strains were set up as controls. All tubes were vulgaris. It was bactericidal at 1× MIC against L. incubated at 37°C for 24 h. The MIC determined was recorded as monocytogenes, S. pyogenes and bacteriostatic to the lowest concentration that inhibits the growth of the bacterial others.
-terpineol was bactericidal against E. coli at both strains. Tween-80 was used as the negative control but showed 1 × MIC and 2 × MIC and only at 2 × MIC for P. vulgaris. minimal effect on the test bacteria. The culture cells without the essential oils were used as the negative control with no effect. The compound was bacteriostatic at both 1 × MIC and 2 × MIC against L. monocytogenes and S. pyogenes. Eugenol on the other hand demonstrated bactericidal Protein leakage assay activity against the entire test bacteria at 2 × MIC where as, at 1 × MIC it was bactericidal against L. monocyto- Protein content in the supernatant obtained by centrifugation of the genes and S. pyogenes and bacteriostatic against the cell suspension treated with different essential oils components (eugenol,
-terpineol and γ-terpinene) at 1 × MIC and 2 × MIC was other test organisms. All three components of essential measured to determine the leakage of intracellular materials from oils possessed bactericidal and bacteriostatic activities at the cells. The samples were incubated at 37oC for 120 min and at different concentrations. 1282 Afr. J. Biotechnol.

(a) (c)

Control 348 Control )

) 198 L

L 1 x MIC 1x MIC m / m 178

/ 298 2 x MIC 2 x MIC m m 158 a r a r

g 248 g 138 o r o r c i c

i 118 198 M ( M

(

98

e

e 148 g g 78 a a k k a a 58 e 98 l e

l

n i

n 38 e i 48 t e t o

18 r o r P -2 P -2 0 30 60 90 120 150 0 30 60 90 120 Time (min) Time (min) (b) (d) Control Control

) 298 158 )

L 1x MIC 1x MIC L m / m 138 2x MIC /

m 248 2x MIC m a a r

r 118 g g o

r 198 o r c 98 i c i M ( M

148 ( 78 e e g g a a

k 58 k 98 a a e e l l

38 n n i i 48 e e t t 18 o o r r p P -2 -2 0 30 60 90 120 0 30 60 90 120 Time (min) Time(min)

Figure 1. Protein leakage in the test organisms. (a) The effect of eugenol on L. monocytogenes; (b) effect of eugenol on S. pyogenes; (c) effect of eugenol on P. vulgaris; and (d) effect of eugenol on E. coli.

Evaluation of protein leakage and the results evidencing the protein leakage corroborated this hypothesis. Eugenol, γ-terpinene and
-terpineol were assessed for their ability to induce cellular protein leakage in P. vulgaris and E. coli (Gram negatives) as well as L. Lipid leakage assessment monocytogenes and S. pyogenes (Gram positives). The three test components were observed to induce protein leakage in all the test organisms at 1 × MIC and 2 × MIC Eugenol, γ-terpinene and
-terpineol were assessed for leading to incremental concentration of protein in the cell their ability to induce cellular lipid leakage in P. vulgaris free media at different time intervals (Figures 1 - 3) up to and E. coli as well as L. monocytogenes and S. as much as 325 g/ml. Both the Gram negative and pyogenes. This was determined by measuring the Gram positive test bacteria showed a similar trend of amount of lipid leakage after treatment with eugenol, γ- protein leakage when treated with eugenol, γ-terpinene terpinene and
-terpineol for a period of 120 min. The and
-terpineol. Eugenol however, had the highest three test compounds were observed to induce lipid damaging effect on cell walls and caused protein leakage leakage in all the test organisms at 1 × MIC and 2 × MIC in the range of 120 – 325 g/ml at 2 × MIC concentration leading to incremental concentration of lipid in the cell (Figure 1). The protein leakage after treatment with γ- free media at different time interval. Treatment of bacteria terpinene ranged from 60 – 225 g/ml at 2 × MIC. The with eugenol showed lipid leakage ranging from 120 - effect of
-terpineol ranged from 70 – 120 g/ml at 2 × 220 g/ml at 2 × MIC and 80 - 170 g/ml at 1× MIC MIC and 50 – 90 g/ml at 1× MIC against the test (Figure 5). The essential oil constituent γ-terpinene bacteria (Figure 3). Protein leakage could be used as an caused lipid leakage ranging from 150 - 550 g/ml at 2× indicator of the membrane damage caused by chemical MIC (Figure 4).
-terpineol damaged cell walls causing and physical agents. It has been suggested that the lipid leakage between 110 - 450 g/ml at 2 × MIC within a cytoplasmic membrane is also a target for eugenol action period of 120 min (Figure 6).

Oyedemi et al. 1283

(a) (c) 98 Control Control ) 88 88 1x MIC L 1x MIC m ) 78 / L

78 m m 2x MIC 2x MIC / a 68 r m g a 68 r o 58 r g o c i r

c 58 i 48 M (

M

(

48 e 38 e g g a a 38 k 28 k a a e e l l

28 18 n n i i e t e t o 18 8 r o r P 8 P -2 0 30 60 90 120 -2 Time (min) 0 30 60 90 120 Time (min) (d) (b)

) 248 Control 78 Control L ) m

/ 1x MIC L 68 1x MIC m m

/ 198 a 2x MIC r

m 2x MIC a 58 g r o g r o c r

48 i 148 c i M M ( (

38 e e g g

a 98 a

k 28 k a a e l e

18 l n

i n e 48 t i o 8 e r t p o -2 r P -2 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min)

Figure 2. Protein leakage in the test organisms. (a) The effect of γ-terpinene on L. monocytogenes; (b) effect of γ-terpinene on S. pyogenes; (c) effect of γ-terpinene on P. vulgaris; and (d) effect of γ-terpinene on E. coli.

(c) (a)

) 98 Control ) Control L L 78 1x MIC m 88 / m 1x MIC / 2x MIC 68 2x MIC m

m 78 a a r r

58 g g 68 o o r r c c 48 i 58 i M M ( (

48

38 e e g g 38 a

a 28 k k a a 28 e e l

l 18

n 18 n i i e

8 e t t 8 o o r -2 r P P -2 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min) (b) (d) Control ) 98 Control L 88

1 x MIC ) m

88 L 1x MIC / 2 x MIC 78 m m 78 / 2x MIC a r m 68 g a

68 r o r g c

o 58 i

58 r c M i (

48

48 M ( e e g 38

38 g a a k k a

28 a 28 e l e

l

n 18 n i 18 i e e t

8 t o

o 8 r r P

-2 P -2 0 30 60 90 120 0 30 60 90 120 Time (min) Time(min)

Figure 3. Protein leakage in the test organisms. (a) The effect of
-terpineol on L. monocytogenes; (b) effect of
-terpineol on S. pyogenes; (c) effect of
-terpineol on P. vulgaris; and (d) effect of
-terpineol on E. coli. 1284 Afr. J. Biotechnol.

(a) (c)

) 398 Control ) L 348 L Control m MIC / 348 m

/ 1x MIC m 2MIC g 298 a

r 298 2x MIC m g a r o 248 r 248 g c o i r c M 198 i ( 198

M ( e

g 148 148 e a g k a a 98 k

e 98 l a

e d l

i 48 48 p d i i L -2 p i -2 0 30 60 90 120 L 0 30 60 90 120 Time (min) Time (min) (b) (d)

248 Control

) Control 598

L 1x MIC 1x MIC ) L m 2x MIC / 2x MIC 198 m /

m 498 m a r a r g g o r 148 o 398 r c i c i M ( M

( 298

e 98 e g g a a k

k 198 a a e l e

l

48 d d i

i 98 p p i i L L -2 -2 0 30 60 90 120 0 30 60 90 120 Time (min) Time (min)

Figure 4. Lipid leakage in the test organisms. (a) The effect of γ-terpinene on L. monocytogenes; (b) effect of γ-terpinene on S. pyogenes; (c) effect of γ-terpinene on P. vulgaris; and (d) effect of γ-terpinene on E. coli.

(a) (c) ) L 198 Control 248 m ) control

/ 1xMIC L

m 2xMIC 1x MIC m / a

r 198

m 2x MIC g 148 a o r r g c o i r 148 M c i (

98

M ( e

g e a g 98 a k k

a 48 a e l e

l

d i d 48 i p p i -2 i L L 0 30 60 90 120 -2 Time (min) 0 30 60 90 120 Time (min) (b) (d) Control 198 1x MIC 198 Control

2x MIC ) 1x MIC ) L 178 L 2x MIC m / m / 158 m

m 148 a a r

r 138 g g o o r r 118 c c i i M M 98 ( 98 (

e e 78 g g a a k k 58 a a e e l 48 l

38 d d i i p p i i 18 L L -2 -2 0 20 40 60 80 100 120 0 30 60 90 120 Time (min) Time (min)

Figure 5. Lipid leakage in the test organisms. (a) The effect of eugenol on L. monocytogenes; (b) effect of eugenol on S. pyogenes; (c) effect of eugenol on P. vulgaris; and (d) effect of eugenol on E. coli. Oyedemi et al. 1285

(a) (c) Control Control ) 198 118 L 1x MIC ) 1xMIC L m

/ 2x MIC m

/ 2xMIC

m 98 a m

r 148 a r g g o

r 78 o r c i c i M

( 98 M

( 58

e e g g a a k 38 k a 48 a e l e

l

d i d 18 i p p i i

L -2 L 0 30 60 90 120 -2 0 20 40 60 80 100 120 Time (min) Time (min) (b) (d)

248 Control Control ) 498 1x MIC )

1x MIC L L 2xMIC m 448 2x MIC / m /

198 m m

a 398 r a r g

g 348 o o r r

148 c c i 298 i M M ( (

248

e e 198

98 g g a a k k 148 a a e e l l

48 98 d d i i

p 48 p i i L -2 L -2 0 30 60 90 120 0 20 40 60 80 100 120 Time (min) Time (min)

Figure 6. Lipid leakage in the test organisms. (a) The effect of
-terpineol on L. monocytogenes; (b) effect of
-terpineol on S. pyogenes; (c) effect of
-terpineol on P. vulgaris; and (d) effect of
- terpineol on E. coli.

DISCUSSION more susceptible to the time kill study at the same concentration, although the MIC was of closer range. The results of Minimum inhibitory concentration (MIC) However, this may be related to the outer membrane showed that two gram positive bacteria, L. monocyto- composition (Nikaido and Vaara, 1985). genes and S. pyogenes were less sensitive to inhibition Further evaluation was carried out to ascertain the of eugenol,
-terpineol and γ-terpinene at concentration effect of essential components against the bacterial between 0.25 to 0.50% (Table 1) than the two gram membrane components. The result of lipid leakage negative bacteria, P. vulgaris and E. coli at 0.50 to showed that eugenol,
-terpineol, and γ-terpinene were 0.75%. The difference in sensitivity to the essential oils effective against both Gram negative and Gram positive components is supported by other researchers including bacteria. γ-terpinene displayed a stronger effect on the Shelef (1983) and Smith- Palmer et al. (1997). During the lipid component of cell membranes of L. Monocytogenes time–kill test, eugenol and γ-terpinene showed similar and E. coli, at both 1 × MIC and 2 × MIC after 120 min of activity which was higher than
–terpineol at the con- incubation than eugenol and
- terpineol (Figure 4 and 5). centrations tested. E. coli and P. vulgaris seemed to be The effectiveness of γ-terpinene might be the result of its very sensitive to the oil components. Reduction in the phenolic structure which interferes with the lipid bilayer of order of 3 to 8 log10 was obtained after 20 h of the outer membranes (Janssen et al., 1987).
-terpineol incubation at 2 × MIC, with E. coli showing the highest and eugenol showed similar effect on the lipid content of reduction of 7.9 log10 reductions after 20 h of incubation cell membrane of both Gram positive and Gram negative followed by P. vulgaris showed 3 log10 reductions. At 1 × bacteria after 120 min of incubation. All the organisms MIC, four strains of bacteria mentioned above showed 3 tested were very susceptible to the effect of essential oil to 4 log10 reduction of bacterial growth (Table 1). It is not components. The different effects observed could be due known exactly why gram negative bacteria should be to the hydrophobicity of the essential oils components 1286 Afr. J. Biotechnol.

which enable them to partition the lipids of the bacterial REFERENCES cell membrane and mitochondria, disturbing the cell Bradford MM (1976). A rapid and sensitive method for the quantitation structures and rendering them more permeable (Sikkema of microgram quantities of protein utilizing the principle of protein- et al., 1994). dye binding. Anal. Biochem. 72: 248-254. The evaluation of protein leakage of the three essential Irobi ON, Moo-Young M, Anderson WA (1996). Anti-microbial activity of
Annatto extract. Internal J. Pharmacol. 34: 87-90. oil components; eugenol, γ-terpinene and -terpineol Janssen AM, Scheffer JJC, Svendsen A (1987). Antimicrobial activity of showed a strong effect on the protein leakage of both the essential oils. Planta-Med. 5: 365-395. Gram negative and Gram positive bacteria. Eugenol Lattaoui N, Tantaoui-Elaraki A (1994). “Individual and combined displayed a stronger effect on the protein leakage of cell antibacterial activity of the main components of three thyme essential oils. Rivista Italiana EPPOS 13:13-19. membranes of P. vulgaris followed by S. pyogenes and L. Marino M, Bersani C, Comi G (2001). Impedance measurement to study monocytogenes at 2 × MIC (Figure 1a-d), while L. antimicrobial activity of essential oils from Lamiaceae and monocytogenes and S. pyogenes at 1 × MIC showed Compositae, Int. J. Food Microbiol. 67: 187-195. high leakage of protein after 120 min of incubation than γ- Nikaido H, Vaara M (1985). Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49: 1-32. terpinene and
-terpineol (Figures 1 - 3). E. coli and L. Smith-Palmer A, Stewart J, Fyfe L (1998). Antimicrobial properties of monocytogenes treated with γ-terpinene and
-terpineol plant essential oils and essences against five important food-borne respectively at 2 × MIC showed high content of lipid pathogens. Lett. Appl. Microbiol. 26: 118-122. leakage (Figures 2d and 3c). Both the Gram negative and Ultee A, Gorris LG, Smid EJ (1998). Bactericidal action of carvacrol towards the food-borne pathogen Bacillus cereus. J. Appl. Bacteriol. Gram positive test bacteria showed a similar trend of 85: 211-218. protein leakage when treated with eugenol, γ-terpinene van Handel E (1985). Rapid determination of total lipids in mosquitoes. and
-terpineol. Eugenol however, had the highest J. Am. Mosq. Contr. Assc. 1: 302-304. Yaki BH, Zurenko GE (2003). An in vitro time-kill assessment of damaging effect on cell walls and caused protein leakage linezolid and anaerobic bacteria. Anaerobe, 9: 1-3. in the range of 120 – 325 g/ml at 2 × MIC concentration (Figure 1). A much lower protein leakage from L. monocytogenes cells treated with
- terpineol and high protein leakage from P. vulgaris treated with eugenol was observed among the three essential oil components tested (Figure 3a). However, γ-terpinene showed higher effect on the test bacteria than
-terpineol of the bacteria tested. Eugenol was very active despite its relatively low capacity to dissolve in water, which is in agreement with published data (Lattaoui and Tantaoui-Elaraki, 1994). In conclusion, this study showed that essential oils components used in this study had bactericidal effect against the both gram positive and gram negative bacteria by disrupting their outer membrane. An important characteristic is their hydrophobicity, which enable them to partition the lipids of bacteria cell membrane disturbing the cell structure and rendering them more permeable. The present investigation provides support to the effectiveness of antibacterial properties of the essential oils tested. Especially in the light of the current trend in finding alternative remedies against increasing numbers of pathogenic bacteria that are resistant to current antibiotics. However, more studies are still needed to validate the mechanism of action of essential oils components.