A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA CHIMICA 12. 1998________________
linke Barelm an*, C arl H einz H a m a n n *, Sig rid Jan nsen ** E L E C T R O C H E M IC A L O X ID A T IO N
AND A N T IB A C T E R IA L P R O P E R T IE S O F PH E N O L IC C O M P O N E N T S O F E SSE N T IA L O ILS
A systematically related group o f phenolic substances the majority o f which is found in essential oils was investigated for a correlation between their bactericide potential and electrochemical oxidizability. For this purpose we have determined the effective concentrations o f the 50% growth inhibition o f E. coli and the half peak potentials o f the oxidation by cyclovoltammetry in diluted phosphate buffer at glassy carbon for phenol, guajacol, catechol as well as for their p-allyl- and p-propenyl derivatives. We found a relation between the effectivity o f the substances, presented as logarithm o f the reciprocal effective concentration log (ECjo)'1. the logarithm o f the octanol/water distribution coefficient log K„w, and the half peak potential Ep,2 o f the electrochemical oxidation.
INTRO DUCTIO N
Essential oils are volatile plant com ponents w hich may occur in different parts o f plants. Som e essential oils are not only bactericide, but also fungicide. E xperim ents have show n, that phenols are am ong the m ost active antim icrobial com ponents o f the essential oils [1], Little is know n about the m echanism s o f effect on m icroorganism s o f essential oils so far. T euscher et al. [2] assum e that the type o f m echanism depends on their concentrations. In the present paper w e therefore investigate the question of w hether the antim icrobial activity o f phenols correlates w ith their electrochem ical oxidizability and their octanol/w ater distribution coefficients. T he phenols to be studied w ere chosen considering the follow ing three aspects: occurrence in essential oils, strong antim icrobial effect and arrangem ent of system atically related group. This led to phenol, chavicol and anol, to guajacol, eugenol and isoeugenol and to catechol and 4-allylcatechol [3 a-3 h]. T he basis o f a variety o f correlations know n from literature is a structure-effect relationship first published by H ansch [4] (Equation (1) ).
* Carl-von-Ossietzky University Oldenburg, Fachbereich Chemie.
** Fachbereich Biologie D-26111 Oldenburg, Carl-von-Ossietzky Strasse 9 -1 1 , Germany; e-mail: hamann@chemie.uni-oldenburg.de.
log (ECso) ' 1 = - a (log Kow) 2 + b log K„w + log KA + c o + d (1)
w here E C5 0 is the effective concentration causing a 50% inhibition o f a biological process, Kow is the octanol/w ater distribution coefficient, KA is the affinity constant betw een substance and receptor, a is the substituent constant according to Hammet, and a - d are general constants. Equation (1) is based on the follow ing assum ptions:
- the substance reaches the place o f action via diffusion through lipophilic and hydrophilic phases (-a (log KoW) 2 + b log K™),
- an interaction betw een the substance and a receptor takes place (log KA), - a reaction betw een the substance and the receptor leads to the biological effect o f the substance (c a).
In case o f phenols, equation (1) can be sim plyfied. It is known from the literature [5], that the binding force of phenols to bovine serum album ine can be described by equation (2).
log (EC) ' 1 = a log Kow + b (2)
Here, EC is the concentration required to form a com plex in a predeterm ined ratio. T herefore it can be assum ed, that the form ation o f the com plex (log KA) as well as the diffusion o f the substance can be described by the logarithm o f the distribution coefficient. A dditionally, there is a linear relationship betw een the substituent constant 0 and the h alf step potential E ¡/2 o f the electrochem ical oxidation for m- and p-substituted phenols [6]. T he substituent constant may thus be replaced by a param eter o f the electrochem ical oxidation, thus the equation (1) can be transform ed to equation (3).
log ( E C 5 0 ) ' 1 = - a (log KoW) 2 + b log Kow + c E m + d (3)
Finally, in case all log K«w values are either below or above 4.3, according to [7] it may be assum ed, that there is a linear relationship betw een (E C5 0 ) 1 and log K«w
instead o f a parabolic one. Hence w e determ ined EC5 0 values, electrochem ical oxidizability and log KoW values in order to establish a correlation.
M ETH ODS A ND M A TERIALS
1. D eterm ination ofE C so values
D eterm ination o f the effective concentrations leading to a 50% inhibition o f a biological process was done by m eans o f a grow th inhibition test w ith the strain Escherichia coli K12 “ w ild type” ATCC 23716. T he m edium used was the DSM 1
m edium (5 g peptone (M erck, from casein) and 3 g m eat extract (M erck) are added to distilled w ater to yield 1 d m 3) proposed for E. coli by the D eutsche Sam m lung von M ikroorganism en und Z ellkulturen GmbH [8], T he pH value was adjusted to 7.4. The m edium was autoclaved at 393.15 K and 1.2-1.4 bar for 20 m inutes.
The sam ple o f 0.5 c m ’ o f an overnight culture (17 hours, 310.15 K) o f the bacterial strain E. coli K 12 was added to 25 cm3 o f the m edium and incubated at 310.15 K for 3 hours. Subsequently this preculture was diluted to an optical density (percentage o f intensity loss due to turbidity) o f 5% . 0.25 c m ' o f this solution was added to 2.25 cm3 of the m edium (as reference) and to 2.25 cm3 o f the m edium containing a certain concentration o f one o f the eight substances under study (optical density 0.5% ). In addition to four parallel sam ples in both cases a blank (w ithout bacteria) was prepared.
The sam ples were incubated at 310.15 K for four hours on a rotating shaker in the dark, follow ed by chilling in ice water. T he optical density was determ ined using a spectrophotom eter Shim adzu U V -120-01 (w avelength 550 nm, path length 1 cm). T he inhibitory effect on cell grow th was calculated in dependence of the concentration o f the phenolic com pounds from the optical data o f the reference, the sam ples containing the com pounds and the blank, follow ing the procedure described in [9]. In case o f som e com pounds (catechol and 4-allylcatechol) the sam ples show ed different discolouring, depending on presence or absence of bacteria. The optical density corresponding to the blank w as therefore determ ined follow ing centrifugation of the sam ples. In the case o f isoeugenol this was not possible, since it form s an em ulsion due to its low w ater solubility. In this case the blank had to be used. The E C5 0 values needed w ere obtained with the help o f a com puter program [1 0].
2. D eterm ination o f data con cern in g the electrochem ical oxidizability
The electrochem ical oxidizability was studied by cyclovoltam m etry in a buffer solution o f pH 7.4. All m easurem ents w ere perform ed in an usual laboratory H-cell o f 25 cm3 volum e. The electronic equipm ent consisted o f a standard Potentiostat (W enking STP 84) in com bination w ith a C P/M M AC 80 (Spectradata) (program C V 2.0 [1 1 ]).
To obtain reproducible cyclovoltam m ogram s o f the phenolic com pounds (concentration range from 0.025 to 5.0 mmol dm '3) it was necessary to use a highly polished glassy carbon electrode. T he electrode was polished (0.05 mm Alum ina) and a reference cyclovoltam m ogram o f 1.0 mmol dm' 3 hexacyanoferrate (II) was taken before each m easurem ent to ensure identical surface conditions. A ccording to our observations this system is very sensitive to surface changes. T he graphically determ ined [12] h alf peak potential Ep/ 2 of the positive sw eep (rate 0.007 V s"1) was taken as significant.
3. D eterm ination o f log Kuw values
Data for phenol, guajacol and catechol are available from literature 113, 14], T he log Kow values o f the rem aining com pounds were calculated according to 114] em ploying fragm ent constants and factors taking the type o f bonding into account.
4. C hem icals
The follow ing com pounds were purchased from com m ercial sources: phenol (Fluka p. a. > 99.5% ), guajacol (M erck reinst > 99% ), eugenol (Fluka > 99% ), isoeugenol (M erck 98% , 75% trans, 25% cis), catechol (M erck > 99% ).
A sam ple o f chavicol w as m ade available through G ivaudan [15]. A further am ount o f chavicol (98.9% ) was synthesized according to Zem plen and Gerecs [16] via the dem ethylation o f I -m ethoxy-4-allylphenol w ith the use o f methyl m agnesium iodide. Anol (99.7% ) was obtained via the dem ethylation of anethol using potassium hydroxide according to Stoerm er and Kahlert [17].
The 4-AUylcatechol (98.5% ) was prepared via the catechol m onoallyl ether [18] and a C laisen rearrangem ent [19]. T rans isoeugenol (99.1% ) was isolated from the com m ercial isoeugenol via the sodium salt [20]. T he progress o f the reactions and the purity of the com pounds w ere m onitored via thin layer and gas chrom atography. For the buffer solutions (adjustm ent of pH value) potassium dihydrogen phosphate (Janssen C him ica p. a.), disodium hydrogen phosphate (M erck p. a.) and 18 M Q w ater (Serai pro 90 c) were used.
RESULTS A ND DISCUSSION
T able 1 show s the substances in a decreasing order o f effective concentrations o f grow th inhibition, logarithm o f the octanol/w ater distribution coefficient and half peak potential.
In the grow th inhibition test guajacol exhibits an activity sim ilar to that of phenol, w hereas catechol is tw ice as active as phenol. T he p-alkenyl-substituted com pounds are m ore active than the respective parent com pounds. T h e higher activity o f isoeugenol com pared to eugenol corresponds w ith results o f agar diffusion tests for antifungicide effects [21], T he fact that chavicol is m ore active than eugenol against E. coli agrees with results o f another agar diffusion test [22].
T he octanol/w ater distribution coefficients are low est for the parent com pounds. On introduction o f the p-alkenyl substituents the log K<,w value changes by an average o f 1.25.
T a b l e 1 List o f substances in a decreasing order o f effective concentrations o f growth inhibition, logarithm o f
the octanol/water distribution coefficient and half peak potential (mean values).
The phenol coefficients are stated in parentheses in the first column following the substance names. Parent com pounds are printed boldface
ECso/mmol dm 5 log Kuw EpnJ Vvs. NHE
phenol (1.0) anol phenol
12.8 2.78 0.787
guajacol (1.1) isoeugenol chavicol
11.2 2.65 0.685
catechol (2.1) chavicol guajacol
6.1 2.65 0.592
eugenol (7.1) eugenol anol
1.8 2.53 0.555
chavicol (8.6) 4-allylcatechol eugenol
1.5 2.04 0.552
isoeugenol (10.5) phenol isoeugenol
1.2 1.46 0.415
4-allylcatechol (12.1) guajacol catechol
1.0 1.33 0.380
anol (14.4) catechol 4-allylcatechol
0.9 0.85 0.335
T he half peak potentials o f the parent com pounds conform w ith values known from literature [2 3 -2 6 ]. T he effect o f the p-propenyl group on the oxidizability is stronger than the effect o f the p-allyl group. T he effect o f p-substituents on the half peak potential decreases from phenol via guajacol to catechol.
In order to determ ine w hether there is a correlation betw een the antibacterial effect o f phenolic substances and their physicochem ical behaviour - represented by the half peak potential o f the electrochem ical oxidation and by the octanol/w ater distribution coefficient - the values obtained can be inserted in equation (3). Since all values o f log KoW are below 4.3, the equation reads as follow s (considering Ep, 2 instead o f E m ):
log (ECso) ' 1 = a log KoW + b Ep/ 2 + c (4) T he constants w ere adjusted to the data of Table 1 by m ultiple regression [10]. T he result obtained is show n in equation (5). Please note that in equation (5) the concentration has to b e inserted in mol dm' 3 and the potential in Volts versus NHE.
log (EC* , ) ' 1 = 0.559 log Kow - 1.34 E ^ + 2.14 (5) T he correlation coefficient is 0.97. In addition a probability > 99% for the existence o f a correlation is indicated by the so called F-Test. T his test is based on the num ber o f param eters (2) and the num ber o f substances un d er study (8).
The determ ined linear correlation betw een the effectivity o f the substances in the growth inhibition test and the logarithm of the octanol/w ater distribution coefficient agrees w ith data found in literature [7]. T he determ ined slope is within the range to be expected and points to an unspecific action by form ation of com plexes with bacterial proteins. However, the correlation presented w ith Ep,2 is a new finding.
A correlation perform ed, as shown above, allow s the determ ination o f the effectivity of related not investigated substances. For instance, the effectivity of 4-propenylcatechol [27] can be estim ated. Equation (5) leads to a value of 0.87 mmol dm' 3 for EC5 0 using log K<)W = 2.17 (determ ined according to [14]), and Ep/2 = 0.220 V versus N H E (extrapolated from the Epn_ data o f T able 1). Hence 4-propenylcatechol w ould have been most effective.
This work was partially presented as a Poster at the Jahrestagung 1996 der G D C h-Fachgruppe A ngew andte Elektrochem ie, M onheim , Germ any.
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Springer Verlag, Berlin
Imke Barelman, Carl Heinz Hamann, Sigrid Jannsen
ELEK TR OC H EM ISC H E OXIDIERBARK EIT UND ANTIBAK TERIELLE EIGENSCHAFTEN PHENOLISCHER BESTANDTEILE ÄTHERISCHER ÖLE
Eine systematisch zusammenhängende Gruppe phenolischer Substanzen, von denen die Mehrzahl in ätherischen Ölen vorkommt, wurde in bezug auf eine Korrelation zwischen antibak-terieller Wirkung und elektrochemischer Oxidierbarkeit untersucht. Zu diesem Zweck wurden die effektiven Konzentrationen, welche eine 50%ige Hemmung des Wachstums von E. coli hervorrufen, sowie die Halbpeakpotentiale für die Oxidation bestimmt (letzteres mittels Zyklovoltammetrie in wässrigem Phosphatpuffer an Glaskohlenstoff). Eingesetzt wurden Phenol, Guajacol und Brenz-katechin sowie deren p-allyl- und p-propenyl- Abkömmlinge. Wir fanden eine lineare Beziehung zwischen der antibakteriellen Wirkung (dargestellt als Logarithmus der reziproken effektiven Konzentration log (ECjo)1), dem Logarithmus des Oktanol/Wasser-Verteilungskoeffizienten KoW und dem Halbpeakpotential Epn der elektrochemischen Oxidation.
lm ke Barelman, Carl H einz Hamann, Sigrid Jannsen
ELEK TROC HEM IC ZNE UTLENIANIE I W ŁASNOŚCI ANTYBAKTERYJNE FEN OLO W YC H POCHO DNYCH OLEJKÓW ETERYCZNYCH
Opisano wyniki badań zależności między bakteriobójczymi własnościami wybranych fenoli, a ich podatnością na eletrochemiczne utlenianie. Badania elektrochemiczne dziewięciu związków z grupy fenoli prowadzono w układzie woda-oktanol. Wyniki badań wskazują na liniową zależność między własnościami antybakeryjnymi (jako odwrotność logarytmu stężenia efektywnego (EC50)'1, logarytmu współczynnika podziału (Kow) i potencjału półfali (Epa) eletrochemicznego utleniania.
