lournal of Clinical Pharmacy and Therapeutics (1992)17.5 1-54
The antimicrobial action of zinc ion/antioxidant combinations T. J. McCarthy, J. J. Zeelie"and D. J. Krause Pharmacy Department, University of Port Elizabeth, P 0 Box 1600 and *PharmacyDepartment, P E Techikon, Private Bag ~ 6 0 1 1Port ,
Elizabefh6000, South Africa
SUMMARY
MATERIALS A N D METHODS
The potentiation of action of antimicrobial preservatives/antiseptics by, respectively, antioxidants and metal ions has been established. In this investigation the antimicrobial effect of two antioxidants (Butylated hydroxyanisole and Propyl Gallate) and zinc ions, both separately and combined, was determined against three organisms at 3 7OC. With the exception of Escherichia coli at low zinc concentrations, definite potentiation occurred, as reflected by a decrease in killing times.
Materials
The following materials were used in this study. Zinc sulphate (ZnS0,.7H20) as a source of ZN2+ ions; butylated hydroxyanisole (BHA), (2- and 3-tert-butyl4-methoxy phenol); propyl gallate (PG), (propyl-3,4,5trihydroxygenzoate); sodium chloride; and ethanol. All materials were from reputable suppliers. Media
Tryptone Soy Broth was used as the recovery medium, followed by 24-h incubation at 37OC.
INTRODUCTION
The fields of disinfection, antisepsis, and preservation are closely related. Before the plethora of antimicrobial agents became available it was a valid assertion to state that phenol, at concentrations of 3, 1 and 0.5% m/v, respectively, could classify as a disinfectant, antiseptic and a preservative. Today we have over 100 preservatives alone, each with its own spectrum of antimicrobial activity and its own inherent problems. Over the last decade there has been an increasing tendency to employ combinations of such antimicrobial agents, both to broaden the spectrum of activity (Grampositive/Gram-negativelfungal)and to discourage the emergence of adaptive resistance. There has also been a move away from potent synthetic chemical structures to consumer friendly or natural products. Our previous work has centred on the antimicrobial action of combinations of selected preservatives and antioxidants (I),and on combinations of selected preservatives, antiseptics and metal ions (2,3).Here we report on combinations of zinc ions and two antioxidants (Butylated hydroxyanisole and Propyl gallate). These have been examined as preservatives (as all three may be orally ingested) and as potential topical deodorants/ antiseptics. It is the latter aspect which is reported here.
Micro-organisms
Each system was separately challenged with an overnight culture of the following, suitably diluted after reading the absorbance at 600nm to give a final concentration of 1 x lo6 ceIIs/mI final test solution. The following micro-organisms were used: Escherichia coli (NCTC 10418),Staphylococcusaureus (NCTC 6571), Candida albicans (patient isolate). N
Methods
Ethanolic solutions of BHA and PG were prepared and sterilized through sterile 0.22 PM filters. All other ingredients were sterilized by autoclaving at 116OC for 30 min. Suitable dilutions of Znzf, BHA and PG were made into normal saline containing a final calculated concentration of ethanol, 4% v/v, to keep the poorly soluble antioxidants in solution.All work was performed in duplicate under a horizontal laminar airflow cabinet. Contact killing time determinations
Prepared inoculated solutions were kept at 37OC and samples (0.1ml) were transferred via sterile pipette
52
T. 1.McCarthy et al.
'
Table 1. Contact killing times at 37°C of zinc ions against three micro-
Time (h)
Zn2 Micro-organism
(pg/ml)
S. aureus
5 10 50
E. coli
5 10 SO
C. albicans
5 10 50
1
5
10
16
18
24
48
organisms in normal saline containing 4%ethanol
+ + + + + + + + + + + + + + +
-
-
+ + + + +
+ + + + +
+ + + + +
+
+
-
-
-
+ + + + +
-
-
-
+ + + +
-
-
+ + + +
-
+ + + +
-
+ =Growth, - =no growth.
Table 2. Contact killing times at 37OC of butylated hydroxyanisole
Time (min) Concentration (%) Micro-organism
BHA
S. aureus
0.038 0040
E. coli
0.038 0.040
C. albicans
0.038 0.040
180 230 250
360
against three micro-organisms in normal saline containing 4%ethanol
+ + + + + +
+
-
-
-
-
++ +- -+ - + - - - -
tips to 2 ml volumes of Tryptone Soy Broth in sterile containers, followed by incubation for 24 h at 37°C. (Tables 1-4). Dilution coefficient (q values) determinations
The q values for the antioxidants were determined (Table 5)using the following formula: log t, - log t, = IogC, -log
270 340
c,
where C, and C, are the first and second concentrations of the test substance; and t, and t, are the killing times of C, and C,, respectively.
+ =Growth, - =no growth. RESULTS A N D DISCUSSION
It can be seen from Table 1 that Zn2+ ions have little effect on microbials except at a concentration of 50pg/d. The same effect has been observed for Minimum Inhibitory Concentrations (MIC) when used alone (2). Furthermore, Zn2+ ions have little effect against the troublesome Psedornonas aeruginosa. Tables 2 and 3, respectively, reflect the killing times of low concentrations of BHA and PG. Although these killing times would be considered as satisfactory and even desirable in topical cosmetic and pharmaceutical formulations, they would not be considered adequate in a clinical situation as a topical antiseptic. Combinations of Zn2+ ions with each of the antioxidants has, however, shown promising potentiation for S. aurew and C.
Antimicrobial action of zinc ion Table 3. Contact killing times at 37OC of propyl gallate against three micro-organisms in normal saline containing 4%ethanol
+ =Growth,
53
Time (min) Micro-organism
Concentration (Pg)
S. aureus
019 0.20
E. coli
019 0.20
C. albicans
0.19 0.20
- = no growth.
Table 4. Effect at 37'C of added zinc ions on the contact killing times of 004% butylated hydroxyanisole and 0.2% propyl gallate against three micro-organismsin normal saline containing 4%ethanol
Micro-organism
Added ZnZ+ (pg/ml) BHA
S. aureus
0
PG
5 10 50
180 70-90 60-70 20-30
220 130 80-90 50
E. coli
0 5 10 50
250 250-270 300 160-170
130 150 150-170 60-80
C. albicans
0 5 10 50
230 15 9 6
240 160 130 60-80
130
190 220
240
Micro-organism
BHA
PG
S. aureus E. colt C. albicans
7.7 7.3 77
7.1 8.1 6-7
330
+ + + + + + + + - - -
albicans. With the latter the addition of only 10 p g / d of ZnZ+ ions to 004% BHA reduced the killing time from 230 to 9 min (Table 4). Propyl gallate in general is less active than BHA but enhanced activity is observed when Zn2+ ions are added. In the case of E. coli Table 4 shows that with only 50 yg/ml an improved killing time is observed for both BHA and PG, with 5 and 10 p g / d actually delaying the killing time. Isobolograms plotted for the minimum lethal concentrations of Zn2+ ions and BHA or PG, respectively, showed definite antagonism for BHA and slight antagonism between the median range of ZnZ+ions and PG against E. coli. Table 5 shows that the antioxidants possess high q values. As shown by the formula Cq x t = k, an increase in concentration of an antimicrobial with a high q value can dramatically reduce the killing time. For example, if phenol (q= 6) at 1% concentration kills an inoculum in 100min, then doubling the concentration should reduce If the phenol should the killing time to 1.56 min (100/26). be decreased to 0.5% by complexation however, (sorption into plastic or rubber, volatization, etc), then the x 106(h)= 100). killing time lengthens to 4.5 days: Thiscan give rise to contaminated products, nosocomial infections and adaptive resistance. Fortunately the QACs have q values of I, and chlorhexidine has an q value of 2 so that dilution will not profoundly affect these classes of antimicrobial agents. We have previously determined bronopol and germall I1 as having values of 0-92and 0.96, respectively, against E. coli (4, and benzoic acid as 1.75, however, the aromatic alcohols benzyl alcohol and phenethyl alcohol gave figures of 7 and 9, respectively (4, so that the compounds, along with the phenolics, need to be carefully monitored for inactivation/dilution effects.
(i6
Table 5. A summary of the q values at 37'C for butylated hydroxyanisole and propyl gallate against three microorganisms in normal saline containing 4%ethanol
310
54
T. 1.McCarthy et al.
This preliminary work indicates potentially useful synergism between the consumer friendly antioxidants BHA and PG and Zn2+ ions at low levels suitable for foodstuffs and oral pharmaceuticals. At the concentrations employed here, however, the killing rate does not indicate any beneficial clinical advantage over existing preparations combined with ZnZ+ions at levels below 50 pg/ml.
REFERENCES 1. Zeelie JJ, McCarthy TJ. (1985)The antimicrobial activities of selected preservatives in the presence of phenolic
antioxidants. South African Pharmacy Journal, 52(4), 161-163. 2. McCarthy TJ. (1985)Metal ions as microbial inhibitors. Cosmetics and Toiletries, 100,69-72. 3. McCarthy TJ, Zeelie JJ.(1989)The effect of zinc ions on
antimicrobial activity of selected preservatives. Journal of Pharmacy and Pharmacology, 41s' 114P. 4. Hurwitz SJ, McCarthy TJ. (1986)2, 3, 5-Triphenyltetrazolium chloride as a novel tool in germicide dynamics. Journalof Pharmaceutical Sciences, 7 5 , 912-916. 5 . Hunvitz SJ, McCarthy TJ. (1987)The effect of pH and concentration on the rated of kill of benzoic acid solutions against E. coli. Journal of Clinical Pharmacy and Therapeutics, 12,107-115.
The antimicrobial action of zinc ion/antioxidant combinations T. J. McCarthy, J. J. Zeelie"and D. J. Krause Pharmacy Department, University of Port Elizabeth, P 0 Box 1600 and *PharmacyDepartment, P E Techikon, Private Bag ~ 6 0 1 1Port ,
Elizabefh6000, South Africa
SUMMARY
MATERIALS A N D METHODS
The potentiation of action of antimicrobial preservatives/antiseptics by, respectively, antioxidants and metal ions has been established. In this investigation the antimicrobial effect of two antioxidants (Butylated hydroxyanisole and Propyl Gallate) and zinc ions, both separately and combined, was determined against three organisms at 3 7OC. With the exception of Escherichia coli at low zinc concentrations, definite potentiation occurred, as reflected by a decrease in killing times.
Materials
The following materials were used in this study. Zinc sulphate (ZnS0,.7H20) as a source of ZN2+ ions; butylated hydroxyanisole (BHA), (2- and 3-tert-butyl4-methoxy phenol); propyl gallate (PG), (propyl-3,4,5trihydroxygenzoate); sodium chloride; and ethanol. All materials were from reputable suppliers. Media
Tryptone Soy Broth was used as the recovery medium, followed by 24-h incubation at 37OC.
INTRODUCTION
The fields of disinfection, antisepsis, and preservation are closely related. Before the plethora of antimicrobial agents became available it was a valid assertion to state that phenol, at concentrations of 3, 1 and 0.5% m/v, respectively, could classify as a disinfectant, antiseptic and a preservative. Today we have over 100 preservatives alone, each with its own spectrum of antimicrobial activity and its own inherent problems. Over the last decade there has been an increasing tendency to employ combinations of such antimicrobial agents, both to broaden the spectrum of activity (Grampositive/Gram-negativelfungal)and to discourage the emergence of adaptive resistance. There has also been a move away from potent synthetic chemical structures to consumer friendly or natural products. Our previous work has centred on the antimicrobial action of combinations of selected preservatives and antioxidants (I),and on combinations of selected preservatives, antiseptics and metal ions (2,3).Here we report on combinations of zinc ions and two antioxidants (Butylated hydroxyanisole and Propyl gallate). These have been examined as preservatives (as all three may be orally ingested) and as potential topical deodorants/ antiseptics. It is the latter aspect which is reported here.
Micro-organisms
Each system was separately challenged with an overnight culture of the following, suitably diluted after reading the absorbance at 600nm to give a final concentration of 1 x lo6 ceIIs/mI final test solution. The following micro-organisms were used: Escherichia coli (NCTC 10418),Staphylococcusaureus (NCTC 6571), Candida albicans (patient isolate). N
Methods
Ethanolic solutions of BHA and PG were prepared and sterilized through sterile 0.22 PM filters. All other ingredients were sterilized by autoclaving at 116OC for 30 min. Suitable dilutions of Znzf, BHA and PG were made into normal saline containing a final calculated concentration of ethanol, 4% v/v, to keep the poorly soluble antioxidants in solution.All work was performed in duplicate under a horizontal laminar airflow cabinet. Contact killing time determinations
Prepared inoculated solutions were kept at 37OC and samples (0.1ml) were transferred via sterile pipette
52
T. 1.McCarthy et al.
'
Table 1. Contact killing times at 37°C of zinc ions against three micro-
Time (h)
Zn2 Micro-organism
(pg/ml)
S. aureus
5 10 50
E. coli
5 10 SO
C. albicans
5 10 50
1
5
10
16
18
24
48
organisms in normal saline containing 4%ethanol
+ + + + + + + + + + + + + + +
-
-
+ + + + +
+ + + + +
+ + + + +
+
+
-
-
-
+ + + + +
-
-
-
+ + + +
-
-
+ + + +
-
+ + + +
-
+ =Growth, - =no growth.
Table 2. Contact killing times at 37OC of butylated hydroxyanisole
Time (min) Concentration (%) Micro-organism
BHA
S. aureus
0.038 0040
E. coli
0.038 0.040
C. albicans
0.038 0.040
180 230 250
360
against three micro-organisms in normal saline containing 4%ethanol
+ + + + + +
+
-
-
-
-
++ +- -+ - + - - - -
tips to 2 ml volumes of Tryptone Soy Broth in sterile containers, followed by incubation for 24 h at 37°C. (Tables 1-4). Dilution coefficient (q values) determinations
The q values for the antioxidants were determined (Table 5)using the following formula: log t, - log t, = IogC, -log
270 340
c,
where C, and C, are the first and second concentrations of the test substance; and t, and t, are the killing times of C, and C,, respectively.
+ =Growth, - =no growth. RESULTS A N D DISCUSSION
It can be seen from Table 1 that Zn2+ ions have little effect on microbials except at a concentration of 50pg/d. The same effect has been observed for Minimum Inhibitory Concentrations (MIC) when used alone (2). Furthermore, Zn2+ ions have little effect against the troublesome Psedornonas aeruginosa. Tables 2 and 3, respectively, reflect the killing times of low concentrations of BHA and PG. Although these killing times would be considered as satisfactory and even desirable in topical cosmetic and pharmaceutical formulations, they would not be considered adequate in a clinical situation as a topical antiseptic. Combinations of Zn2+ ions with each of the antioxidants has, however, shown promising potentiation for S. aurew and C.
Antimicrobial action of zinc ion Table 3. Contact killing times at 37OC of propyl gallate against three micro-organisms in normal saline containing 4%ethanol
+ =Growth,
53
Time (min) Micro-organism
Concentration (Pg)
S. aureus
019 0.20
E. coli
019 0.20
C. albicans
0.19 0.20
- = no growth.
Table 4. Effect at 37'C of added zinc ions on the contact killing times of 004% butylated hydroxyanisole and 0.2% propyl gallate against three micro-organismsin normal saline containing 4%ethanol
Micro-organism
Added ZnZ+ (pg/ml) BHA
S. aureus
0
PG
5 10 50
180 70-90 60-70 20-30
220 130 80-90 50
E. coli
0 5 10 50
250 250-270 300 160-170
130 150 150-170 60-80
C. albicans
0 5 10 50
230 15 9 6
240 160 130 60-80
130
190 220
240
Micro-organism
BHA
PG
S. aureus E. colt C. albicans
7.7 7.3 77
7.1 8.1 6-7
330
+ + + + + + + + - - -
albicans. With the latter the addition of only 10 p g / d of ZnZ+ ions to 004% BHA reduced the killing time from 230 to 9 min (Table 4). Propyl gallate in general is less active than BHA but enhanced activity is observed when Zn2+ ions are added. In the case of E. coli Table 4 shows that with only 50 yg/ml an improved killing time is observed for both BHA and PG, with 5 and 10 p g / d actually delaying the killing time. Isobolograms plotted for the minimum lethal concentrations of Zn2+ ions and BHA or PG, respectively, showed definite antagonism for BHA and slight antagonism between the median range of ZnZ+ions and PG against E. coli. Table 5 shows that the antioxidants possess high q values. As shown by the formula Cq x t = k, an increase in concentration of an antimicrobial with a high q value can dramatically reduce the killing time. For example, if phenol (q= 6) at 1% concentration kills an inoculum in 100min, then doubling the concentration should reduce If the phenol should the killing time to 1.56 min (100/26). be decreased to 0.5% by complexation however, (sorption into plastic or rubber, volatization, etc), then the x 106(h)= 100). killing time lengthens to 4.5 days: Thiscan give rise to contaminated products, nosocomial infections and adaptive resistance. Fortunately the QACs have q values of I, and chlorhexidine has an q value of 2 so that dilution will not profoundly affect these classes of antimicrobial agents. We have previously determined bronopol and germall I1 as having values of 0-92and 0.96, respectively, against E. coli (4, and benzoic acid as 1.75, however, the aromatic alcohols benzyl alcohol and phenethyl alcohol gave figures of 7 and 9, respectively (4, so that the compounds, along with the phenolics, need to be carefully monitored for inactivation/dilution effects.
(i6
Table 5. A summary of the q values at 37'C for butylated hydroxyanisole and propyl gallate against three microorganisms in normal saline containing 4%ethanol
310
54
T. 1.McCarthy et al.
This preliminary work indicates potentially useful synergism between the consumer friendly antioxidants BHA and PG and Zn2+ ions at low levels suitable for foodstuffs and oral pharmaceuticals. At the concentrations employed here, however, the killing rate does not indicate any beneficial clinical advantage over existing preparations combined with ZnZ+ions at levels below 50 pg/ml.
REFERENCES 1. Zeelie JJ, McCarthy TJ. (1985)The antimicrobial activities of selected preservatives in the presence of phenolic
antioxidants. South African Pharmacy Journal, 52(4), 161-163. 2. McCarthy TJ. (1985)Metal ions as microbial inhibitors. Cosmetics and Toiletries, 100,69-72. 3. McCarthy TJ, Zeelie JJ.(1989)The effect of zinc ions on
antimicrobial activity of selected preservatives. Journal of Pharmacy and Pharmacology, 41s' 114P. 4. Hurwitz SJ, McCarthy TJ. (1986)2, 3, 5-Triphenyltetrazolium chloride as a novel tool in germicide dynamics. Journalof Pharmaceutical Sciences, 7 5 , 912-916. 5 . Hunvitz SJ, McCarthy TJ. (1987)The effect of pH and concentration on the rated of kill of benzoic acid solutions against E. coli. Journal of Clinical Pharmacy and Therapeutics, 12,107-115.
