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 *Pharmacy Department, P E Techikon, Private Bag
`~ 6 0 1 1 , Port Elizabefh 6000, South Africa
`
`SUMMARY
`The potentiation of action of antimicrobial
`preservatives/antiseptics by, respectively, anti-
`oxidants 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 or-
`ganisms at 3 7OC. With the exception of Escheri-
`chia coli at low zinc concentrations, definite
`potentiation occurred, as reflected by a decrease
`in killing times.
`
`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 preserva-
`tives 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 (Gram-
`positive/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 preser-
`vatives, 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.
`
`MATERIALS A N D METHODS
`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-butyl-
`4-methoxy phenol); propyl gallate (PG), (propyl-3,4,5-
`trihydroxygenzoate); 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.
`
`Micro-organisms
`Each system was separately challenged with an over-
`night culture of the following, suitably diluted after
`reading the absorbance at 600nm to give a final
`concentration of N 1 x lo6 ceIIs/mI final test solution.
`The following micro-organisms were used: Escherichia
`coli (NCTC 10418), Staphylococcus aureus (NCTC 65 71),
`Candida albicans (patient isolate).
`
`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 con-
`centration of ethanol, 4% v/v, to keep the poorly soluble
`antioxidants in solution. All work was performed in dupli-
`cate under a horizontal laminar airflow cabinet.
`
`Contact killing time determinations
`Prepared inoculated solutions were kept at 37OC and
`samples (0.1 ml) were transferred via sterile pipette
`
`ALCON 2046
`Apotex Corp. v. Alcon Research, Ltd.
`Case IPR2013-00428
`
`

`

`52 T. 1. McCarthy et al.
`
`Micro-organism
`
`Zn2 '
`(pg/ml)
`
`S. aureus
`
`E. coli
`
`C. albicans
`
`5
`10
`50
`
`5
`10
`SO
`
`5
`10
`50
`
`Time (h)
`
`Table 1. Contact killing times at
`37°C of zinc ions against three micro-
`organisms in normal saline containing
`4%ethanol
`1
`5
`10
`16
`18
`24
`48
`+ + + + + + +
`+ + + + + + +
`+ -
`-
`-
`-
`-
`-
`
`+ + + + + + +
`+ + + + + + +
`+ + + + -
`-
`-
`
`+ + + + + + +
`+ + + + + + +
`+ + -
`-
`-
`-
`-
`
`
`+ =Growth, - =no growth.
`
`Micro-organism
`
`Concentration (%)
`BHA
`
`180 230 250 270 340 360
`
`Time (min)
`
`Table 2. Contact killing times at
`37OC of butylated hydroxyanisole
`against three micro-organisms in
`normal saline containing 4% ethanol
`
`S. aureus
`
`E. coli
`
`C. albicans
`
`0.038
`0040
`
`0.038
`0.040
`
`0.038
`0.040
`
`+ + + + + -
`+
`+
`-
`-
`-
`-
`+ + + + -
`-
`+ -
`-
`-
`-
`
`-
`
`
`
`
`
`
`
`
`+ =Growth, - =no growth.
`
`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 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.
`
`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 formu-
`lations, 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.
`
`

`

`Table 3. Contact killing times at
`37OC of propyl gallate against three
`micro-organisms in normal saline
`containing 4% ethanol
`
`Micro-organism
`
`Concentration
`(Pg)
`
`Time (min)
`
`130 190 220 240 310 330
`
`Antimicrobial action of zinc ion 53
`
`S. aureus
`
`E. coli
`
`C. albicans
`
`0 1 9
`0.20
`
`0 1 9
`0.20
`
`0.19
`0.20
`
`+ + + + + -
`+ + + -
`-
`-
`
`
`
`
`
`+ =Growth, - = 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-organisms in normal saline
`containing 4% ethanol
`
`Micro-organism
`
`Added ZnZ+
`(pg/ml)
`
`BHA
`
`PG
`
`S. aureus
`
`E. coli
`
`C. albicans
`
`0
`5
`10
`50
`
`0
`5
`10
`50
`
`0
`5
`10
`50
`
`180
`70-90
`60-70
`20-30
`
`220
`130
`80-90
`50
`
`250
`250-270
`300
`160-170
`
`130
`150
`150-170
`60-80
`
`230
`15
`9
`6
`
`240
`160
`130
`60-80
`
`Table 5. A summary of the q values at 37'C for butylated
`hydroxyanisole and propyl gallate against three micro-
`organisms in normal saline containing 4% ethanol
`
`Micro-organism
`
`S. aureus
`E. colt
`C. albicans
`
`BHA
`
`7.7
`7.3
`7 7
`
`PG
`
`7.1
`8.1
`6-7
`
`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
`100 min, then doubling the concentration should reduce
`the killing time to 1.56 min (100/26). If the phenol should
`be decreased to 0.5% by complexation however, (sorp-
`tion into plastic or rubber, volatization, etc), then the
`killing time lengthens to 4.5 days: (i6 x 106(h) = 100).
`This can 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 pre-
`viously determined bronopol and germall I1 as having
`values of 0-92 and 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.
`
`

`

`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-Triphenyltetra-
`zolium chloride as a novel tool in germicide dynamics.
`Journal of Pharmaceutical Sciences, 75, 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.
`
`