synthesis and antimicrobial characteristics of a novel biocide
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Biocontrol Science, 2004, Vol. 9, No. 4, 95-103
Original
Synthesis and Antimicrobial Characteristics of a Novel
Biocide, 4,4 - (1,6-Dioxyhexamethylene)bis-
(1-alkylpyridinium halide)
TADAO YABUHARA, TAKUYA MAEDA, HIDEAKI NAGAMUNE,
AND HIROKI KOURAI*
Department of Biological Science and Technology, Faculty of Engineering,
The University of Tokushima, Minamijosanjima-cho, Tokushima 770-8506, Japan
Received 10 June 2004/Accepted 6 August 2004
We synthesized a new bis-quaternary ammonium compound (bis-QAC), 4,4 - (1,6-
dioxyhexamethylene) bis (1-alkylpyridinium halide) s (4DOBP-6-n-X) (alkyl chain length, n = 6,
8, 10, 12, 14, 16 and 18, X = Br or I) that has a symmetrical dimeric structure, and is composed
of two alkylpyridinium halides connected with a dioxyhexamethylene. We examined the rela-
tionship of the hydrophobicity and antimicrobial activity using 4DOBP-6-n-X. The influences of
pH, temperature, and hemolytic activity were then measured using 4,4 - (1,6-dioxyhexame-
thylene)bis (octylpyridinium bromide) (4DOBP-6-8-Br) which showed a superior antimicrobial
activity among the 4DOBP-6-n-X series. The following three chemical compounds were used
as a comparison: 4,4 - (1 ,6-dithiohexamethylene)bis(octylpyridinium bromide) (4DTBP-6-8-Br)
which is considered as an excellent antimicrobial agent based on a previous report,
alkylbenzyldimethyl ammonium chloride (BAC) and 2- (4 -thiazolyl)benzimida-zole (TBZ). In
the results of the experiment, 4DOBP-6-8-Br exhibited a wide antimicrobial spectrum, and had
a strong activity against bacteria and fungi comparable to 4DTBP-6-8-Br. The hemolytic ac-
tivities of 4DOBP-6-8-Br and 4DTBP-6-8-Br were almost same, and lower than that of BAC.
4DTBP-6-8-Br had some defects in that it colored metals or generated a bad smell and a haz-
ardous gas; however, in 4DOBP-6-n-X, these disadvantages were not present. 4DOBP-6-n-X
seems to be an environmentally friendly antibacterial agent.
Key words : Quaternary ammonium compound/Novel biocide/Antimicrobial activity/4,4 -(1,6-
dioxyhexamethylene)bis(1-alkylpyridinium halide) .
INTRODUCTION
Quaternary ammonium compounds (QACs) have
been commonly used for paints, water treatment, tex-
tiles, in the food industry and the hospital, and for
medical and domestic use because QACs have a
relatively low toxicity and wide-ranging antimicrobial
spectrum.
For the first time, Domagk disclosed the antimicro-
bial activity of the long-chain QACs such as
alkylbenzyl dimethyl ammonium chloride (BAC)
(Domagk, 1935), a so-called mono-QAC which con-
sists of only one quaternary ammonium group.
Numerous studies on their synthesis and antimi-
crobial characteristics are currently underway. In or-
der to improve the antimicrobial activity of the mono-
QACs, bis-QACs, which consist of two symmetric
quaternary ammonium groups, were synthesized. For
example, N,N,N ,N -tetraalkyl-N,N -bis (alkylbenzyl) alkylbenzyN,
N -2-butynylene-1,4-bis (ammonium halides) [Benne-
ville, P. L. and Bock L.H. (1950) U. S. Patent, No.
* Corresponding author
. Tel : +81-88-656-7408, Fax : +
81-88-656-9148.
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96 T. YABUHARA ET AL.
2,525, 778], the salts of decamethylene-bis-4-
aminoquinaldinium (Babbs et al., 1956) and bis-
QACs derived from bis-(2-dimethylaminoethyl)
glutarate (Pavlikova-Moricka et al., 1994) have been
prepared. Subsequently, a series of modified bis-
QACs as potential antimicrobial agents, 4,4 -( a, co
polymethylenedithio) bis (1-alkylpyridinium halide) s
(4DTBP-m-n-X) (Okazaki et al., 1997), 4,4 -(1,6-he-
xamethylene-dioxydicarbonyl) bis (1-alkylpyridinium
iodide)s (4DOCBP-6-n) (Maeda et al., 1999a), 5,5 -
[2,2 -(tetramethylenedicarbonyldioxy) diethyl]bis (3-
al kyl-4-methylthiazolyium iodide) s (5DEBT-4-n)
(Maeda et al., 1999b), and N,N -hexamethylene-
bis(4-carbamoyl-1-decylpyridinium bromide) (D-38)
(Yoshida et al., 2000) were synthesized and studied.
Previous studies revealed that QACs act on the cell
wall and have a direct or indirect lethal effect on the
cell. In addition, it was proved that the factors which
control their antimicrobial activity are molecular
hydrophobicity (Kourai et al., 1983b and 1995),
adsorbability (Kourai et al., 1983a), and the electron
density of the ammonium nitrogen atom (Maeda et
al., 1996; Okazaki et al., 1996) or bacterioclastic ac-
tivity (Kourai et al., 1994).
We considered that the 4DTBP-m-n-X was the best
QAC as described below. In previous studies, it was
demonstrated that it had wide antimicrobial effects
against both bacteria and fungi, and these activities
were stronger than those of the typical bactericide
BAC or the popularly used fungicide TBZ. Moreover,
the bactericidal activities of 4DTBP-m-n-X were not
affected by the length of the hydrophobic alkyl chain.
They were comparatively uninfluenced by environ-
mental conditions, such as pH or temperature.
However, at the same time, the 4DTBP-m-n-X has
some defects as follows. First, in an aqueous solution
it colors metal surfaces. Also, hazardous gases, such
as hydrogen sulfide, sulfur dioxide, etc., are gener-
ated from 4DTBP-m-n-X, not only during its manufac-
turing process but also during its decomposition.
These phenomena would cause the contamination of
the environment. In order to modify these disadvan-
tages in this study, we presented 4DOBP-6-n-X as a
new compound, in which the sulfur atom of 4DTBP-
m-n-X is substituted by oxygen. Therefore, if the
antimicrobial activities of 4DOBP-6-n-X are better
than those of 4DTBP-m-n-X, it will be a novel
antimicrobial agent that is environmentally friendly.
MATERIALS AND METHODS
Chemicals
The procedure to synthesize 4DOBP-6-n-X is
shown in Fig.1. The abbreviations, n and X, indicate
the carbon number of the alkyl chain and the halogen
atom, respectively. 4,4 -(1,6-Dithiohexamethy-
lene)bis(1-octylpyridinium bromide) (4DTBP-6-8-Br)
was chosen as a representative of 4DTBP-m-n-X. All
chemicals used to synthesize the compounds were
reagent grade commercial materials and used without
further purification. The synthesis of 4DOBP-6-n-X
was more difficult than that of 4DTBP-6-n-X since it is
dependent on its chemical reactivity, i.e., the less re-
active 4-hydroxypyridine does react with the
alkylhalide directly. Therefore, it was synthesized by
a method different (Fig.1) from that described in the
previous report (Okazaki, 1997). The reaction of 4-
hydroxypyridine with sodium in N,N-dimethylformal-
FIG. 1. Procedure to synthesize 4,4 -(1,6-dioxyhexamethylene)bis(1-alkylpyridinium halide)s (4DOBP-6n-X)
.
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NOVEL ANTIMICROBIAL AGENT 97
dehyde provided the alcoholate. It was further con-
verted into 4DODP-6 by the addition of 1,6-
hexamethylenedibromide. The final products,
4DOBP-6-n-X s, were obtained in the reaction of
4DODP-6 and the corresponding n-alkyl halides, in
anhydrous ethanol at 80•Ž, and 80MPa for 72 h. The
products were recrystallized from ethanol-diethyl
ether to give a white powder and dried in a vacuum.
The procedures to synthesize 4DTBP-6-n-X were
traced by thin-layer chromatography (Merck silica gel
60 F254 plates and Merck RP-18 F254S plates, thickness
0.25mm, Merck Japan, Ltd., Tokyo). The final prod-
ucts were identified by elemental analyses (Yanaco
CHN Corder, MT-5, Yanagimoto, Kyoto) and proton
nuclear magnetic resonance (NMR, 400MHz, JEM-
EX 400, JEOL, Tokyo) spectra data. The melting
points were measured with a melting point apparatus
(Mitamura Riken Kogyo Inc., Tokyo). The decompo-
sition points were recorded on a TG-DTA apparatus
(PTC-10A, Rigaku, Tokyo). The solubility was judged
by visual observation after agitation for 24 h at 25•Ž.
4DTBP-6-8-Br was synthesized by a method de-
scribed in a previous report (Okazaki et al., 1997).
BAC (alkyl =CnH2n+1 n= 8 -18) was purchased from
Kanto Chemical Co., Inc. (Tokyo). TBZ was obtained
from San-ai Oil, Ltd. (Tokyo).
Molecular hydrophobicity
The chromatographic RM value, related to the loga-
rithm of the partition coefficient, can be used to esti-
mate the molecular hydrophobicities of the QACs
(Franke, 1984).
Therefore, in this study, thin layer partition chroma-
tography (DC-Fertigplatten, RP-18, F254S Merck
Japan Ltd.) was used to determine the RM values, and
then these were employed to estimate their molecular
hydrophobicities. The thin layer chromatography was
performed using an acetonitrile-ethyl alcohol (10 :10)
solvent system at 30•Ž for 30min. The flow rates of
the samples were determined under irradiation with
UV light. The RM value is defined as
RM = log ((1/Rf) -1)
where a is the flow ra te of a specific 4DOBP-6-n-X.
Microbes, cultivation and preparation
Escherichia coli IFO 12713 was used in these ex-
periments, unless otherwise noted. Bacteria and fungi
were basically prepared by a method described in
previous report (Maeda et al., 1999b). The bacterial
cell suspensions were also prepared by a previously
reported method (Kourai et al., 1991) and kept in ice-
cold water for 2h before use. Nutrient broth medium
was purchased from Becton, Dickinson and Co.
Nutrient agar medium was obtained from OXOID Ltd.
Sabouraud agar and potato dextrose agar medium
was bought from Nissui Pharmaceutical Co., Ltd.
Antibacterial activity
The minimum bactericidal concentration (MBC)
and minimum inhibitory concentration (MIC) of the
antimicrobials were measured by the method previ-
ously described (Okazaki et al., 1997). Bacteriostatic
activity and bactericidal activity were defined as log
MIC-1 and log MBC-1.
Erythrocyte preparation and hemolytic activity
These procedures were carried out using a modi-
fied method of Fogt et al. (1995). Human blood
TABLE 1. 1H-NMR spectra of 4DOBP-6-n-X.
a1H
-NMR spectra were measured in Materials and Methods using tetramethylsilane as an internal standard as described.
b See the text for the full name of this compound
.
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98 T. YABUHARA ET AL.
drawn from the first author was washed three times in
a phosphate-buffered saline (PBS) by centrifugation
at 1500 •~g for 5 min at 4•Ž to obtain pure erythro-
cytes. PBS (pH7.4) consisted of 6.78g NaCI, 1.42g
Na2HPO4, and 0.4g KH2PO4 in 1 liter of distilled water.
The erythrocytes were then suspended at a concen-
tration of 2 •~ 109cells/ml in the PBS by determining
the number of erythrocytes with a hemocytometer
(Thoma Blutkorperzahlapparat, Tiefe 0.1mm,
1/400qmm). The antimicrobial agents like QACs
were stepwise diluted by PBS to a final volume of 1
ml. Ten ƒÊ l of the erythrocyte suspension (2 •~ 109
cells/ml) was then added to the above solution and
the final erythrocyte concentration was 2 •~ 107cells/-
ml. The erythrocyte solutions were next incubated at
37•Ž for 30min. Following the incubation, the solu-
tions were centrifuged at 600 •~g for 5 min at 4•Ž, and
the percent hemolysis was determined by comparing
the absorbance at 540 nm of the supernatants with
that of a control sample totally hemolyzed with dis-
tilled water :
hemolysis ( ) = C (AQAC - A PBS / (A control - A PBS) ) •~
100
where APBss the absorbance of the supernatant with
PBS. The hemolytic activity, HC50, s the concentra-
tion which induces a 50 release of hemoglobin from
the erythrocytes, which was determined from the plot
of the percentage of hemolysis versus the concentra-
tions of the QACs.
RESULTS AND DISCUSSION
Chemical structures and properties of 4DOBP-6-
n-X
In Table' 1, the chemical structure assignments for
the compounds are based on their 1H-NMR. For ex-
ample, in 4DOBP-6-8-Br, the signal at 0.90ppm (t, J
=7.0Hz
, 6H) indicated the terminal methyl protons of
the alkyl chain which is connected to the ammonium
nitrogen. The signals of 1.30-1.40ppm (m, 20H),
1.41-1.54 (m, 4H), 1.88ppm (m, 4H) and 1.99ppm
(m, 4H) were assigned to the methylene protons of
the alkyl and alkylene chains. As for the methylene
protons bonded to the ether oxygen or the ammonium
TABLE 2. Elemental analyses of 4DOBP-6-n-X.
a Numbers indicate
.
TABLE 3. Yields and physical properties of 4DOBP-6-n-X.
a The yields of 4DOBP
-6-n-X were calculated as 4DOBP-6-n-X versus 4
-hydroxypyridine
.
b Decomposition was observed at two points by TG/DTA
, heating rate 10•Ž/min, under a nitrogen flow of 200ml/min, ref-
erence, Al2O3.
che solubilities of 4DOBP-6-n-X were measured in water at 25•Ž and indicated as weight
.
d Values are mean•}S
.D., obtained from three independent experiments.
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NOVEL ANTIMICROBIAL AGENT 99
nitrogen, the chemical shifts were seen at 4.36ppm
(t,=6.5Hz, 4H) or 4.46ppm (t,J=7.6Hz, 4H). These
chemical shifts were confirmed by a two dimensional
proton-carbon NMR correlation spectroscopy (C-H
cosy) experiment (data not shown). The signals at
7.51ppm (d, J=7.6Hz, 4H) and 8.75ppm (d, J=7.6Hz,
4H) were assigned to the protons of the pyridine ring.
The described 1H-NMR data are consistent with the
proposed chemical structure of 4DOBP-6-8-Br. Data
for the other compounds are shown in Table 1. It is
worth noting that there was a remarkable difference
between the chemical shifts of the methylene proton
which is bonded to the oxygen or sulfur. That is, in
4DOBP-6-8-Br and 4DTBP-6-8-Br, they were respec-
tively 4.36 ppm (t, J=6.5Hz, 4H) and 3.24ppm (t, J
=7.4Hz
, 4H), while the other data did not show a sig-
nificant difference between the two compounds. It
can be presumed that the differences are attributable
to the gap between the electronegativity of the oxy-
gen and sulfur atoms.
In Table 2, the data for the elemental analyses were
all within •}0.3 of the calculated values. In Table 3,
the yields, the melting points, the decomposition
points and the solubilities are listed. As a result, all of
the synthesized 4DOBP-6-n-X compounds had the
FIG. 2. Relationship between molecular hydrophobicity
(RM) and the bacteriostatic activity (log MIC-1) of 4DOBP-6-
n-Br. The unit of MIC is molarity(M). Symbols : •œ,
Escherichia cob' IFO 12713 ; •, Staphylococcus aureus
IFO 12732. The number under the symbol in the figure ex-
presses n. Solid lines represent the equations : E. coil IFO
12713, log MIC-1 =4.96-0.756 RM-1.86(RM)2 ; (coefficient
of correlation r=0.791); St. aureus IFO 12732, log MIC-1
=6
.05+0.992 RM-4.80 (RM)2 ; (r= 0.932) .
FIG. 3. Relationship between molecular hydrophobicity
(RM) and the bactericidal activity (log MBC-1) of 4DOBP-6-
n-Br. The unit of MIC is molarity(M). Symbols : •œ,
Escherichia coil IFO 12713 ; •, Staphylococcus aureus
IFO 12732. The number under the symbol in the figure ex-
presses n. Solid lines represent the equations : E. cob' IFO
12713, log MBC-1=5.10+0.952 RM -.03(RM)2 ; (coefficient
of correlation r=0.506); St. aureus IFO 12732, log MBC-1
=5.90+3.20 RM-3.30 (RM)2 ;r= 0.860) .
proposed chemical structures, and the analytical data
indicated sufficient purity for the following investiga-
tion of their antimicrobial characteristics.
Molecular hydrophobicity of 4DOBP-6-n-X
In Table 3, an approximately linear relationship ex-
ists between the alkyl chain length of 4DOBP-6-n-Br
and its molecular hydrophobicity. Therefore, the
change in its molecular hydrophobicity will be de-
pendent on the length of the alkyl group of the pyri-
dine.
Effect of molecular hydrophobicity on the
antimicrobial activity
As described above, the attachments of the differ-
ent lengths of alkyl groups changed the molecular
hydrophobicity of the QACs, and then the effects of
the molecular hydrophobicities (RM) on their
antimicrobial activities were evaluated. The RM data of
4DOBP-6-n-Br are shown in Table 1, and plotted ver-
sus their antimicrobial activities. The resulting curves
were parabolic against E. coil IFO 12713 and
Staphylococcus aureus IFO 12732, but the coeffi-
cients of the second-order equation of the
bacteriostatic activities (footnote in Fig. 2) were
greater than for their bactericidal activities (footnote
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100 T. YABUHARA ET AL.
in Fig.3). Though there were differences in the values
of the antimicrobial activity against the two bacteria,
similar behavior was seen. As indicated in Fig. 2, the
bacteriostatic activities of 4DOBP-6-n-Br were in-
creased when n=6 increased to n=8, and slightly de-
creased when n=8 increased to n=18. On the other
hand, in Fig. 3, their bactericidal activities were simi-
larly increased with the range from n=6 to n=8, but
FIG. 4. Effect of temperature A) and pH B) on the bacte-
ricidal activity log MBC-1) of 4DOBP-6-8-Br and BAC
against Escherichia coil IFO 12713. The unit of MIC is
molarity M). Symbols:•œ4DOBP-6-8-Br;•, BAC.
they were almost constant over n=8. These results
suggest that the hydrophobicities of 4DOBP-6-n-Br
have a smaller influence on the bactericidal activities
than on the bacteriostatic activities. Except for n=6,
the tendency of 4DOBP-6-n-Br n=8 to n=18) resem-
bled the results previously reported for 4DTBP-6-n-X
n=8 to n=18) Maeda et al., 1998). The reason why
the bacteriostatic activities of 4DOBP-6-n-Br n=8 to
18) declined in Fig. 2 compared with the bactericidal
activities is thought to be due to the difference in the
measurement system. The bactericidal activity is
based on the interaction between the antimicrobial
agent and bacterial cell in the water without other ma-
terials, while the bacteriostatic activity reflects the in-
teraction in the nutrient medium containing
hydrophobic materials such as peptone. Therefore, it
is implied that the hydrophobic materials in the me-
dium interact with the hydrophobic group of the
antimicrobial molecule such as the alkyl chain, inhibit
the contact with the bacterial cell surface, and even-
tually reduce the antibacterial activity. The result of
our experiment clearly showed that the antimicrobial
activities of 4DOBP-6-n-X were a little affected by the
molecular hydrophobicities like in the case of 4DTBP-
6-n-X.
Effects of pH and temperature on bactericidal
activity
The effects of pH on the bactericidal activity of
4DOBP-6-8-Br and BAC were measured using 0.05M
phosphate buffer pH=5, 6, 7, 8 and 8.7) against E.
coil IFO 12713.
In Fig. 4, the bactericidal activity of 4DOBP-6-8-Br
was constant and higher than that of BAC in the
range from pH5 to pH8.7. On the other hand, the bac-
TABLE 4. Minimum inhibitory concentrations MICs) of 4DOBP-6-8-X, 4DTBP-6-8-Br and BAC against bacteria.
a Alkylbenzyldimethyl ammonium chloride
.
b Values are mean •}S
.D., obtained from three independent experiments.
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NOVEL ANTIMICROBIAL AGENT 101
TABLE 5. Minimum Inhibitory concentrations (MICs) of 4DOBP-6-8-Br, 4DTBP-6-8-Br and TBZ against fungi
.
a Values are mean•}S
.D., obtained from three independent experiments.
b Obtained from National Institute of Health Sciences
.
TABLE 6. Hemolytic concentrations (NC50)a of 4DOBP-6-8-Br, 4DTBP-6-8-Br and BAC against human red blood cells.
a HC
50 (ƒÊM) was measured by a dilution method using a phosphate-buffered saline at 37•Ž for 30 min and was the con-
centration of QACs inducing 50 hemolysis.
b Values are mean•}S
.D., obtained from three independent experiments.
tericidal activity of BAC decreased under acidic con-
ditions. It is known that the antimicrobial activity of a
mono-QAC, such as BAC, is lower in acid conditions
than in alkali (Kourai et al., 1994) which is one of its
disadvantages. To the contrary, the bactericidal activ-
ity of 4DOBP-6-8-Br was not dependent on pH, which
is due to its dimeric structure.
ubsequently, the effects of temperature on the an-
tibacterial activities of 4DOBP-6-8-Br and BAC were
examined at various temperatures (10, 20, 30, and
40•Ž) against E coil IFO 12713. The other conditions
of this antibacterial activity test were the same as in
the method described above in Materials and
Methods. There were no effects of the bacterial
growth on this result, since the MBCs were measured
in a suspension without nutrient substances for 30
min. As can be seen in Fig. 4, the bactericidal activity
of 4DOBP-6-8-Br was almost equal, which was higher
than that of BAC, at temperatures from 10•Ž to 40•Ž.
However, the activity of BAC was slightly influenced
by the temperature, and decreased by lowering the
temperature. In a previous study, the antimicrobial ac-
tivity of bis-QACs was hardly influenced by tempera-
ture, but that of mono-QACs was. The compounds
with other alkyl groups showed a similar tendency
(Maeda et al., 1999b).
In this experiment, the activities of 4DOBP-6-8-Br
and BAC had a tendency to resemble those in a pre-
vious study. Generally, an increase in the temperature
tends to enhance the bactericidal activities of the
QACs, because the temperature is closely related to
the fluidity of the bacterial cell membrane and most
QACs interact with the membrane to produce a bac-
tericidal action. In this study, the bactericidal activity
of BAC was influenced by the fluidity of the bacterial
cell membrane but that of 4DOBP-6-8-Br was not. It
could be suggested that this was caused by the
dimeric structure of 4DOBP-6-n-X.
As described above, the bactericidal activities of
4DOBP-6-8-Br were not affected by conditions of pH
or temperature. These are considered the merits of
4DOBP-6-n-X when it is used as an antimicrobial
agent.
Antimicrobial spectra of 4DOBP-6-8-Br, 4DTBP-6-
8-Br, BAC and TBZ
The antibacterial spectra of 4DOBP-6-8-Br,
4DTBP-6-8-Br and BAC were examined using a MIC
measurement system against the gram-positive bac-
teria (6strains) and gram-negative bacteria
(7strains) of stationary-phase cells, and fungi
(8strains). The MIC measurement system is an
antimicrobial evaluation which is carried out in a nutri-
ent broth composed of organic materials. The anti-
bacterial and the antifungal spectra of their
compounds are summarized in Tables 4 and 5. Both
4DOBP-6-8-Br and 4DTBP-6-8-Br had wide
antimicrobial spectra and exhibited a higher
antimicrobial activity than BAC or TBZ. In Table 4,
the difference in the antimicrobial activity could not
be found between 4DOBP-6-8-Br and 4DOBP-6-8-I.
Therefore, 4DOBP-6-8-Br was used for the experi-
ment.
In this experiment, 4DOBP-6-8-Br and 4DTBP-6-8-
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102 T. YABUHARA ET AL.
Br had higher activities against gram-positive bacteria
than against the gram-negative, as in the case of the
general QACs. Moreover, remarkable differences in
their activities were seen between bis-QACs
(4DOBP-6-8-Br, 4DTBP-6-8-Br) and a mono-QAC
(BAC) against gram-negative bacteria, though not so
against the gram-positive bacteria. This would be due
to the fact that the cell surface of the gram-positive
bacteria is more hydrophobic than that of gram-
negative bacteria (Kourai et al.,1989). In conclusion,
it was implied that the excellent antimicrobial activi-
ties of 4DOBP-6-n-X are due to its unique dimeric
structure.
Hemolytic activity of 4DOBP-6-8-Br, 4DTBP-6-8-
Br and BAC
The hemolytic activity of 4DOBP-6-8-Br was evalu-
ated and compared to that of 4DTBP-6-8-Br and a
commercial BAC on human red cells (Table 6). Tac
was assumed as one of the indications of acute
cytotoxicity using the data of HC50 and MIC. It is
thought that the safety of an antibacterial agent also
becomes high as the value of Tac becomes large. The
Tao is defined as
Tac=Hac/BMIC
where Hacand BMIC are the aforedescribed HC50 value
ƒÊ) and MIC(ƒÊM).
From the above formula using MIC of Table 4 and
HC50 of Table 6, Tac of 4DOBP-6-8-Br was 138 to
1.44,and that of BAC was 72.7 to 0.48. Therefore, it is
presumed that the safety of 4DOBP-6-8-Br is higher
than BAC. On the other hand, the hemolytic activity
HC50 value of 4DTBP-6-8-Br was almost same as that
of 4DOBP-6-8-Br.
It is possible that the differences between the
HC50 values of 4DOBP-6-8-Br and BAC could be at-
tributed to their chemical structures. Further work is
now in progress to confirm this mechanism.
In conclusion, in this study, it was proved that
4DOBP-6-n-X had excellent antimicrobial activities
and safety regarding hemolysis. Moreover, we cannot
expect 4DOBP-6-n-X to generate a foul odor and
harmful gases due to its chemical structure nor to
have the property of coloring metals. Hence 4DOBP-
6-n-X would be a new excellent antimicrobial agent
that is environmentally friendly. Further investigations
to characterize 4DOBP-6-n-X are now in progress.
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