Skip Navigation

Journal of Antimicrobial Chemotherapy 2006 58(4):773-777; doi:10.1093/jac/dkl336
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Henriques, A.
Right arrow Articles by Burton, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Henriques, A.
Right arrow Articles by Burton, N.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Free radical production and quenching in honeys with wound healing potential

Ana Henriques1, Simon Jackson2, Rose Cooper1 and Neil Burton1,*

1 School of Applied Sciences, University of Wales Institute Cardiff Llandaf Campus, Western Avenue, Cardiff CF5 2YB, UK 2 Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK

*Corresponding author. Tel: +44-2920-416845; Fax: +44-2920-416982; E-mail: nburton{at}uwic.ac.uk

Received 18 May 2006; returned 20 July 2006; revised 24 July 2006; accepted 25 July 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objectives: Honey-impregnated wound dressings are now available on drug tariff in the UK, though the modes of action of honeys with antibacterial and wound healing properties are not entirely clear. The action of some but not all of these honeys is linked to the production of hydrogen peroxide on dilution of the honey with wound exudate. The present study investigates both free radical production and the antioxidant potential of some honeys, properties which may have a role to play in wound healing.

Methods: Free radical production and quenching of three honey types (manuka, antibacterial but non-peroxide-producing; pasture, antibacterial peroxide-producing; commercial heat processed, non-antibacterial) was investigated by electron paramagnetic resonance (EPR) spectroscopy; quenching was also examined using a superoxide quenching assay.

Results: All honeys tested had antioxidant potential, with manuka able to completely quench added radicals within 5 min of spiking. Only the peroxide-producing honey (pasture PS9) was found to form radicals on dilution.

Conclusions: The ability to modulate production and quenching of free radicals may contribute to the demonstrated ability of some honeys to help in resolving the state of inflammation typifying chronic wounds.

Keywords: manuka , antioxidants , antibacterial , EPR spectroscopy


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Traditionally, carefully selected honey has been used for the treatment and prophylaxis of many disorders.1 Honey is enjoying a renaissance in wound care1 because it offers broad-spectrum antimicrobial properties and it promotes rapid wound healing,2 though the mechanisms by which these effects are achieved have not been fully elucidated.

Honey is a complex product; it has low water activity and acidity, and high sugar content but this does not contribute significantly to the antibacterial activity of those honeys recommended for clinical use.3,4 Some of these honeys become more potent upon dilution due to production of hydrogen peroxide generated by the action of glucose oxidase deposited in honey by bees.5 Addition of catalase to diluted honeys of this type destroys the activity of hydrogen peroxide6 and reduces or eliminates antibacterial activity. A survey of New Zealand honeys demonstrated that antimicrobial activity of a few honeys (mostly derived from Leptospermum scoparium, or manuka shrub) was retained on dilution in the presence of catalase.7 These honeys therefore rely on components other than hydrogen peroxide for their potency on dilution, and these have been postulated to be phytochemicals8 (though it has been suggested that peroxide still has a role, even in these ‘non-peroxide’ honeys).9

The production of peroxide could lead to the formation of free radicals such as hydroxyl and superoxide. While these may play a part in antimicrobial activity (they are produced by human immune cells), they are potentially damaging to tissues and are also produced by bacteria to enable easier access to human cells.10–12

In addition to antibacterial activity, honeys are known to have antioxidant capacity, which may act to modulate production of free radicals.8 The object of the present study was to investigate free radical production of peroxide and non-peroxide honeys using electron paramagnetic resonance (EPR) spectroscopy and to assess their antioxidant potential using EPR and a superoxide quenching assay.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Three samples of natural honey were used. A clear, uncrystallized honey (Gale's) obtained from a supermarket was included to represent a heat-processed honey (heat processing destroys antimicrobial activity), an antibacterial peroxide-generating honey from mixed floral source (pasture PS9) and monofloral non-peroxide-generating antibacterial manuka M109 honey (not heat processed). An artificial honey that reflected the main components of honey was prepared by dissolving 1.5 g of sucrose, 7.5 g of maltose, 40.5 g of fructose and 33.5 g of glucose in 17 mL of deionized water.4 This solution was included in the study to evaluate the contribution of the predominant sugars and water to the assayed activities.

EPR spectroscopy was employed in two series of experiments. First the detection of hydroxyl radical (OH•) formation from hydrogen peroxide that may be produced in the diluted honey samples, second to test for any antioxidant activity in the honey by quenching free radicals deliberately introduced into diluted honey samples.13 To detect highly reactive radicals, such as OH•, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Sigma) was used. This compound reacts with OH• to form a stable paramagnetic species (the ‘spin-adduct’ DMPO-OH•) that gives a characteristic 1:2:2:1 quartet of lines in the EPR spectrum.

To determine the potential for OH• production by the different honey solutions, Fe2+ was added to the honey to generate OH• by Fenton chemistry from any peroxide present. As a positive control for trapping this radical, Fe2+ was used to generate the hydroxyl radical from H2O2.

To determine OH• production 40 µL of 0.1 M DMPO was added to 100 µL of a fresh 50% (w/v) solution of honey in an Eppendorf tube and mixed gently by pipetting. To initiate the reaction, 20 µL of 0.05 M FeSO4 was added and the solution immediately pipetted into a sample tube and placed in an EPR spectrometer (Varian E104 operating at 9.2 GHz microwave frequency and 20 mW power) and EPR spectra recorded. Typical spectrometer conditions were 100 Gauss (G) scan range, 120 s scan time and 1.0 G field modulation. Experiments were repeated in the presence of 1 mg/mL catalase.

In experiments to detect antioxidant activity (radical quenching) by EPR, the honey samples were also ‘spiked' with 30 µL of 30% (v/v) H2O2 prior to addition of the FeSO4 and the decay time of the resultant EPR spectrum was used to determine the OH• radical ‘quenching' or antioxidant potential of each honey.

To further investigate antioxidant activity a superoxide quenching assay was used; superoxide radicals were generated by the xanthine/xanthine oxidase (X/XO) system following a method described previously.14,15 Each assay contained 290 µL of 5 mM xanthine (sodium salt), 100 µL of the test solution (positive control, 50% honey solution or negative control) and 100 µL of 1 mM lucigenin. Positive control was superoxide dismutase (Sigma) 1000 U/mL, and negative control was sterile deionized water. After mixing, 150 µL of this mixture was transferred to wells in a 96-well microtitre plate using at least five replicates per sample. To each well 10 µL of xanthine oxidase (0.5 U/mL) was added and the plate was incubated for 15 min in the dark. The plate was read in a luminomenter (MLX Microtiter Plate Luminometer; Dynex Technologies) and results expressed in relative light units (RLU).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
Hydroxyl radical formation

A total of 58 EPR spectra were examined. Figure 1 shows the EPR spectra for positive and negative controls and Figure 2 the spectra for pasture honey with and without the addition of catalase. The only honey observed to generate radicals was the pasture honey.


Figure 1
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. (a) EPR spectrum of the OH• radical trapped with DMPO from the Fenton reaction (positive control). (b) The same as in (a) but without Fe2+ or H2O2 (negative control).

 


Figure 2
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. (a) Typical EPR spectrum obtained with a 50% (w/v) solution of pasture honey in the presence of FeSO4 and DMPO. (b) Same as (a) but with the addition of 1 mg/mL catalase.

 
Antioxidant activity by EPR

Figure 3 shows the EPR spectra for manuka honey before adding Fe2+, H2O2 and DMPO (a), immediately after addition (b) and 5 min after addition (c), demonstrating complete quenching of the OH• spectrum. Speed of signal quenching varied between honeys. Gale's also produced complete quenching 5 min after the additions; artificial honey (Figure 4) produced only partial quenching after 5 min. The pasture honey was the slowest quencher as it actively produces radicals on dilution (Figure 2), but after 1 h the EPR spectrum was reduced to a similar level to that generated by the addition of catalase in the experiments to determine OH• production (Figure 2).


Figure 3
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Antioxidant capacity of manuka honey. (a) Typical EPR spectra obtained for 50% (w/v) manuka honey, and for 50% (w/v) manuka honey incubated with H2O2 + FeSO4 and DMPO, measured at (b) time 0 and (c) after 5 min following addition of H2O2.

 


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Antioxidant capacity of artificial honey. (a) Typical EPR spectra obtained for 50% (w/v) artificial honey, and for 50% (w/v) artificial honey incubated with H2O2 + FeSO4 and DMPO, measured at (b) time 0 and (c) after 5 min following addition of H2O2.

 
Antioxidant activity by superoxide quenching assay

Variation in quenching activity was detected (Figure 5), with manuka honey similar to the positive control, followed by pasture, Gale's, artificial and negative control.


Figure 5
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Ability of different honey solutions to quench superoxide radical generated from the xanthine/xanthine oxidase system as determined by chemiluminescence. Positive quenching control was superoxide dismutase (1000 U/mL) and negative quenching control was distilled water.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
The only honey found to generate radicals in the present study was pasture PS9. In those honeys that do produce peroxide, bactericidal activity could be due to hydroxyl and possibly other radical products. In support of this statement catalase treatment reduces antibacterial activity,7 also in this study spectra of the hydroxyl radical and other radical species were observed on activation of pasture honey with ferrous ion. The additional radicals could not be unequivocally identified; their spectra suggest that they may arise from secondary reactions between hydroxyl radicals and organic compounds present in the honey. These secondary organic radicals may also have antibacterial properties. Any antibacterial properties of a peroxide-generating honey will thus be a balance between the opposing activities of radical generation and quenching.

It has been shown for peroxide honeys that the optimal dilution at which the honey will produce maximal amounts of hydrogen peroxide is between 40% and 60%.16 The fact that no radical production was seen from manuka honey on dilution to 50% confirms this honey as non-peroxide, relying on other mechanisms for its antibacterial and wound healing properties, contrary to previous suggestion.9 These mechanisms are as yet unknown, but are possibly derived from the range of phytochemicals present in manuka honey.

All the honeys tested by EPR and superoxide quenching assay had some antioxidant capability when compared with controls. The manuka honey, in particular, showed complete quenching of the EPR signal generated by peroxide within 5 min. Even the pasture honey, which actively generates radicals, showed quenching after 1 h. Manuka honey has been shown to promote synthesis of cytokines by monocytes that have the potential to mediate the immune response.2,17 In responding to challenges such as infected wounds, neutrophils and macrophages utilize the presence of free radicals such as superoxide and hydroxyl to modulate the activity of other cells such as monocytes and platelets. These in turn produce specific cytokines to signal activation of other cells.18 However, in a prolonged insult, such as a chronically infected wound, such stimuli may give rise to an excessive response and the ability to dampen free radicals may therefore contribute to the complex interaction that helps to resolve the state of chronic inflammation typifying these wounds.

When Pseudomonas aeruginosa invades human tissue it uses free radical formation as a means to enhance invasion, and this can lead to cellular damage.10 Manuka honey is bactericidal to P. aeruginosa in vitro and has been suggested as a topical treatment for wounds infected with this bacterium.3 The present study shows that the quenching properties of manuka honey in particular, which have been attributed to methyl syringate,13 could minimize the invasive effects of such an infection as well as help to resolve chronic inflammation.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
A. H., S. J. and N. B. have no conflicts of interest. R. C. has received grants from the BSAC, the SGM, the European Wound Management Association and the University of Waikato (in collaboration with the US National honey board). Sponsorship to attend scientific meetings has been received from Capilano and Comvita, consultancy has been undertaken from Brightwake Ltd, Medlock Medical and Medihoney, and remuneration for presentations has been received from the Tissue Viability Society and beekeeping organizations.


   Acknowledgements
 
We are grateful to Professor P. C. Molan of Waikato University for the gift of the honey. A. H. was supported by a studentship from UWIC.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 Dixon B. (2003) Bacteria can't resist honey. Lancet Infect Dis 3:116.[CrossRef][Web of Science][Medline]

2 Tonks AJ, Cooper RA, Jones KP, et al. (2003) Honey stimulates inflammatory cytokine production from monocytes. Cytokine 21:242–7.[CrossRef][Web of Science][Medline]

3 Cooper RA, Halas E, Molan PC. (2002) The efficacy of honey in inhibiting strains of Pseudomonas aeruginosa from infected burns. J Burn Care Rehabil 23:366–70.[CrossRef][Web of Science][Medline]

4 Cooper RA, Molan PC, Harding KG. (2002) The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. J Appl Microbiol 93:857–63.[CrossRef][Medline]

5 White JW Jr, Subers MH, Schepartz AI. (1963) The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochim Biophys Acta 73:57–70.[Medline]

6 Adcock D. (1963) The effect of catalase on the inhibine and the peroxide values of various honeys. J Apic Res 1:38–40.

7 Molan PC. (1992) The antibacterial activity of honey. 1. The nature of the antibacterial activity. Bee World 73:5–28.[Web of Science]

8 Gheldof N, Wang XH, Engeseth NJ. (2002) Identification of antioxidant components of honeys from various floral sources. J Agric Food Chem 50:5870–7.[CrossRef][Web of Science][Medline]

9 Weston RJ. (2000) The contribution of catalase and other natural products to the antibacterial activity of honey: a review. Food Chemistry 71:235–9.[CrossRef]

10 Britigan BE, Roeder TL, Rasmussen GT, et al. (1992) Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury. J Clin Invest 90:2187–96.[Web of Science][Medline]

11 Nunoshiba T, DeRojas-Walker T, Tannenbaum SR, et al. (1995) Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages. Infect Immun 63:794–8.[Abstract]

12 Wang Y, Aisen P, Casadevall A. (1995) Cryptococcus neoformans melanin and virulence: mechanism of action. Infect Immun 63:3131–6.[Abstract]

13 Inoue K, Murayama S, Seshimo F, et al. (2005) Identification of phenolic compound in manuka honey as specific superoxide anion radical scavenger using electron spin resonance (ESR) and liquid chromatography with coulometric array detection. J Sci Food Agric 85:872–8.[CrossRef][Web of Science]

14 Valentao P, Fernandes E, Carvalho F, et al. (2002) Studies on the antioxidant activity of Lippia citriodora infusion: scavenging effect on superoxide radical, hydroxyl radical and hypochlorous acid. Biol Pharm Bull 25:1324–7.[CrossRef][Web of Science][Medline]

15 Valentao P, Fernandes E, Carvalho F, et al. (2002) Antioxidant activity of Hypericum androsaemum infusion: scavenging activity against superoxide radical, hydroxyl radical and hypochlorous acid. Biol Pharm Bull 25:1320–3.[CrossRef][Web of Science][Medline]

16 Bang LM, Buntting C, Molan P. (2003) The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing. J Altern Complement Med 9:267–73.[CrossRef][Web of Science][Medline]

17 Tonks A, Cooper RA, Price AJ, et al. (2001) Stimulation of TNF-{alpha} release in monocytes by honey. Cytokine 14:240–2.[CrossRef][Web of Science][Medline]

18 Burdon RH. (1995) Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 18:775–94.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Evid. Based Med.Home page
D. G Armstrong
Manuka honey improved wound healing in patients with sloughy venous leg ulcers
Evid. Based Med., October 1, 2009; 14(5): 148 - 148.
[Full Text] [PDF]

Home page
 INT J LOW EXTREM WOUNDSHome page
W. J. Lindblad
Review Paper: Considerations for Determining if a Natural Product Is an Effective Wound-Healing Agent
International Journal of Lower Extremity Wounds, June 1, 2008; 7(2): 75 - 81.
[Abstract] [PDF]

Home page
 Arch Pediatr Adolesc MedHome page
I. M. Paul, J. Beiler, A. McMonagle, M. L. Shaffer, L. Duda, and C. M. Berlin Jr
Effect of Honey, Dextromethorphan, and No Treatment on Nocturnal Cough and Sleep Quality for Coughing Children and Their Parents
Arch Pediatr Adolesc Med, December 1, 2007; 161(12): 1140 - 1146.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. J. Tonks, E. Dudley, N. G. Porter, J. Parton, J. Brazier, E. L. Smith, and A. Tonks
A 5.8-kDa component of manuka honey stimulates immune cells via TLR4
J. Leukoc. Biol., November 1, 2007; 82(5): 1147 - 1155.
[Abstract] [Full Text] [PDF]

Home page
 INT J LOW EXTREM WOUNDSHome page
B. Shivananda Nayak, S. Sivachandra Raju, F.A. Orette, and A.V. Chalapathi Rao
Effects of Hibiscus rosa sinensis L (Malvaceae) on Wound Healing Activity: A Preclinical Study in a Sprague Dawley Rat
International Journal of Lower Extremity Wounds, June 1, 2007; 6(2): 76 - 81.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Henriques, A.
Right arrow Articles by Burton, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Henriques, A.
Right arrow Articles by Burton, N.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?