- Email: [email protected]
Treatment of non-healing leg ulcers with honeydew honey
Journal of Tissue Viability (2014) 23, 94e97
www.elsevier.com/locate/jtv
Clinical study
Treatment of non-healing leg ulcers with honeydew honey Alexander Mayer a,1, Viliam Slezak b,1, Peter Takac c,d, Juraj Olejnik a, Juraj Majtan c,e,* a
Department of Surgery, University Hospital and Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia b University Hospital of the Merciful Brothers, Bratislava, Slovakia c Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia d Scientica, s.r.o., Bratislava, Slovakia e Department of Microbiology, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia
KEYWORDS Honey; Wound; Treatment
Abstract Background: Honey is used as a traditional medicine for centuries by different cultures for the treatment of various disorders. However, not all honeys exhibit equal antimicrobial potency and only a few meet the criteria for clinical usage. Aim: The aim of the study was to determine clinical efficacy of sterilised honeydew honey in the treatment of the lower leg ulcers in 25 patients. Furthermore, we evaluated honey acceptability of patients in terms of pain and overall satisfaction. Methodology: A total of 25 patients with chronic venous leg ulcers were recruited into this study. The 100% g-irradiated sterile honeydew honey was applied onto the cleaned wounds and each wound was assessed at the least two times in for a period of 6 weeks. Results: During the course of treatment, the average wound area of all patients decreased significantly from 51 (3e150) to 22 (0e91) cm2. Eighteen patients (72%) experienced a decrease in reported pain levels while five patients (20%) experienced the same level of pain throughout the study. The overall satisfaction with honey treatment was positive in 80% of patients. Only two patients experienced poor tolerance due to problems at ulcer site related to pain. Conclusion: Based on these findings, honeydew honey has the potential to be one of the medical-grade honeys. ª 2014 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Institute of Zoology, Slovak Academy of Sciences, Dubravska cesta 9, 845 06 Bratislava, Slovakia. Tel.: þ421 2 59302647; fax: þ421 2 59302646. E-mail address: [email protected] (J. Majtan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jtv.2014.08.001 0965-206X/ª 2014 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.
Treatment of non-healing leg ulcers
Introduction Hard-to-heal leg ulcers, or ulcers of a duration longer than 4-weeks and those, which fail to respond to standard care, are the major cause of morbidity in elderly population. Venous or stasis ulcers account for 80%e85% of all leg ulcers [1]. The standard treatment of venous ulcers is the compression of the lower leg by tight bandaging [2] but other treatment options have been proposed including honey therapy. Honey has been used as a traditional medicine for centuries by different cultures for the treatment of various disorders. It offers broad-spectrum antimicrobial properties and promotes rapid wound healing [3]. The antibacterial potential of honey has been considered the exclusive criterion for its wound healing properties. Therefore, antibacterial activity of honey from different floral sources has been intensively studied over the past few decades. Manuka honey, a type of medical-grade honey is the most frequently studied in human clinical trials and claimed to have therapeutic advantages over other honeys. However, some concerns regarding the toxicity of methylglyoxal, and, major antibacterial compound of manuka honey, have been recently expressed [4]. Slovak fir honeydew honey has recently showed excellent antibacterial activity against multi-drug resistant clinical isolates of Stenotrophomonas maltophilia [5] and wound pathogens [6]. It possesses anti-biofilm [6] and anti-MMP-9-activity [7] in vitro. In addition, it has been successfully applied in the treatment of infected gluteofemoral fistulas [8] and as prophylactic agent of endophthalmitis [9]. In this study, we characterised a clinical efficacy of sterilised honeydew honey in the treatment of the lower leg ulcers in 25 patients. Furthermore, we evaluated honey acceptability to patients in terms of pain and overall patient satisfaction.
Patients and methodology A total of 25 patients were recruited for this study. The study protocol was approved by University Hospital ethical committee, and informed consent was obtained from all participants. All patients had chronic venous leg ulcers for more than 12weeks’ duration at the time of presentation, and an ulcer area range of 3e150 cm2. Venous leg ulcers were selected after clinical examination. The baseline demographics of the recruited patients are presented in Table 1. Before the treatment,
95 Table 1
Patient characteristics.
Age (years) Gender Ulcer aetiology Ulcer size (cm2)
Mean Range Male Female Arterial-Venous Mixed Mean Range
79 59e93 12 13 19 (76%) 6 (24%) 51 3e150
ulcers were cleaned with cleansing agents or desloughing materials. The 100% g-irradiated sterile honeydew honey was applied onto the cleaned wounds using sterile spatula, covered by a non-adhering dressing and changed after 1e3-days depending on a wound exudation. Each wound was assessed at the least three times for a period of 3e6 weeks. Primary outcomes such as change in ulcer area, rate of ephithelisation and adverse effects were determined. In addition, all patients were asked to record their subjective feelings (for example, pain level) and overall satisfaction during the honey treatment. Level of pain experienced was assessed as follows: 0 ¼ pain decreased; 1 ¼ pain remained the same; 2 ¼ pain increased. Patients rated the overall satisfaction with the honey treatment on a numerical scale 1e3 (1 ¼ poor, 2 ¼ average, 3 ¼ good).
Results From the twenty-five patients with venous insufficiency or mixed leg ulcers recruited two patients did not complete the study due to problems at ulcer site related to pain. Eighteen patients (72%) experienced a decrease in reported pain levels while five patients (20%) experienced the same level of pain throughout the study (Fig. 1). During the course of treatment, the average wound area of all patients decreased significantly from 51 (3e150) to 22 (0e91) cm2. Honey-induced process of wound healing was also photodocumented in some cases (Fig. 2). Overall tolerance of honey was good after 6 weeks of treatment. In 80% of all assessments, the results were positive; in 12%, there was no change in tolerance; and in 8% there were complaints of poor tolerance (patients dropped out of the study) (Fig. 3).
Discussion In this study, Slovak honeydew honey improved leg ulcer healing within 6 weeks. All patients who
96
Fig. 1 Patients (n ¼ 25) pain evaluation during honey treatment. Level of pain experienced was assessed as follows: 0 ¼ pain decreased; 1 ¼ pain remained the same; 2 ¼ pain increased.
completed study reported overall satisfaction with the honey therapy. Although honey is generally well tolerated, 8% of the patients did not complete the study due to continuous pain. Pain is only known side-effect of using honey (This appears to be due to the acidity of honey, as pain is not experienced when neutralised honey is used): 28% of patients in the largest case series reported pain associated with honey [10]. On the other hand, many authors also documented that most of the patients (60%) experienced a decrease in reported pain levels. This is in accordance with the results of our study where pain was decreased in 72% of the patients. According to a very recent analysis of 25 clinical trials (with a total of 2987 participants) that evaluated the effect of honey on the rate of healing acute wounds (such as burns, lacerations) and chronic wounds (such as skin ulcers, infected
A. Mayer et al. surgical wounds) found that honey dressings do not increase the rates of healing significantly in venous leg ulcers when used as an adjuvant to compression [11]. Nevertheless there are clinical benefits associated with the use of honey in the management of wounds in clinical practice such as reduction in healing duration and treatment costs (consumables and nursing time) and improvement in the quality of life. In addition, authors of above mentioned Cochrane review analysed only studies that report complete wound healing as a primary outcome and did not take into account several clinical studies where wound healing was not achieved but wound size decreased [12]. Limited number of honeys of different botanical and geographical origin is being used clinically. Several other types of honey such as Malaysian tualang honey or Slovak honeydew honey have the potentials being another medical-grade honeys. Honeydew honey may heal wounds through a combination of pathways. It has been shown that honeydew honeys generates higher amounts of hydrogen peroxide than other honeys including manuka honey [13]. Hydrogen peroxide is one of the major antibacterial components and is produced by glucose oxidase e mediated conversion of glucose to gluconic acid under aerobic conditions in diluted honey [14]. Based on several findings [15,16] the overall antibacterial activity of honeys was greatly correlated with H2O2 production in the honeys. Besides the well known honey effects such as osmosis and lowering wound pH, honeydew honey also exhibits a strong antiinflammatory effects partially via inhibition of MMP-9. Recently, it has been postulated that increased levels of pro-inflammatory cytokines as well as the dysregulation of MMP-9 are involved in impaired healing in patients with chronic venous leg ulcers [17]. In particular, the level of MMP-9 remains permanently high and MMP-9 appears to be a major protease responsible for the degradation of matrix and cell growth-promoting agents in chronic wound fluids [18].
Fig. 2 Treatment of leg ulcers with honeydew honey before and after 6 weeks. Honey was sterilized and applied to a depth of 3 mm (20 g of honey to a 10 10 cm area).
Treatment of non-healing leg ulcers
97
References
Fig. 3 The overall patients satisfaction with honey treatment.
According to our very recent study [7], honeydew honey attenuated TNF-a-induced MMP-9 expression in human keratinocytes and suggest that the specific flavonoid content of honey is able to down-regulate the expression of MMP-9. Therefore, the inhibition of MMP-9 secretion depends on the particular flavonoid content of each honey including medical-grade ones.
Conclusions Honeydew honey is a promising wound-healing agent, represents an ideal inexpensive agent that meets all criteria to be therapeutically useful in treating chronic wounds. Thus, honeydew honey has the potential to be one of the medical-grade honeys.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0115-11, and the Operational Program of Research and Development and co-financed with the European Fund for Regional Development (EFRD). Grant: ITMS 26240220020 e “Establishment of biotherapeutic facility and technology proposal for production and development of biodrugs”.
[1] Collins L, Seraj S. Diagnosis and treatment of venous ulcers. Am Fam Physician 2010;81:989e96. [2] Robson MC, Cooper DM, Aslam R, Gould LJ, Harding KG, Margolis DJ, et al. Guidelines for the treatment of venous ulcers. Wound Repair Regen 2006;14:649e62. [3] Molan PC. The evidence supporting the use of honey as a wound dressing. Int J Low Extrem Wounds 2006;5: 40e54. [4] Majtan J. Methylglyoxal-a potential risk factor of manuka honey in healing of diabetic ulcers. Evid Based Complement Alternat Med 2011;2011:295494. [5] Majtan J, Majtanova L, Bohova J, Majtan V. Honeydew honey as a potent antibacterial agent in eradication of multi-drug resistant Stenotrophomonas maltophilia isolates from cancer patients. Phytother Res 2011;25:584e7. [6] Majtan J, Bohova J, Horniackova M, Majtan V. Anti-biofilm activity of natural honey against wound bacteria. In: Majtan J, editor. Honey: current research and clinical applications, vol. 1. New York: Nova Science Publishers, Inc.; 2012. p. 83e106. [7] Majtan J, Bohova J, Garcia-Villalba R, Tomas-Barberan FA, Madakova Z, Majtan T, et al. Fir honeydew honey flavonoids inhibit TNF-a-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing. Arch Dermatol Res 2013;305:619e27. [8] Vlcekova P, Krutakova B, Takac P, Kozanek M, Salus J, Majtan J. Alternative treatment of gluteofemoral fistulas using honey: a case report. Int Wound J 2012;9:100e3. [9] Cernak M, Majtanova N, Cernak A, Majtan J. Honey prophylaxis reduces the risk of endophthalmitis during perioperative period of eye surgery. Phytother Res 2012; 26:613e6. [10] Dunford CE, Hanano R. Acceptability to patients of a honey dressing for non-healing venous leg ulcers. J Wound Care 2004;13:193e7. [11] Jull AB, Walker N, Deshpande S. Honey as a topical treatment for wounds. Cohrane Database Syst Rev 2013;2: CD005083. [12] Cooper R. Impact of honey as a topical treatment for wounds remains unclear. Evidence-based Med 2014;19:11. [13] Bucekova M, Valachova I, Kohutova L, Prochazka E, Klaudiny J, Majtan J. Honeybee glucose oxidase e its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys. Naturwissenschaften 2014;101:661e70. [14] White JW, Subers MH, Schepartz AI. The identification of inhibine, the antibacterial factor in honey, as hydrogenperoxide and its origin in a honey glucose-oxidase system. Biochim Biophys Acta 1963;73:57e70. [15] Brudzynski K. Effect of hydrogen peroxide on antibacterial activities of Canadian honeys. Can J Microbiol 2006;52: 1228e37. [16] Chen C, Campbell LT, Blair SE, Carter DA. The effect of standard heat and filtration processing procedures on antimicrobial activity and hydrogen peroxide levels in honey. Front Microbiol 2012;3:1e8. [17] Serra R, Buffone G, Falcone D, Molinari V, Scaramuzzino M, Gallelli L, et al. Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin. Wound Repair Regen 2013; 21:395e401. [18] Moor AN, Vachon DJ, Gould LJ. Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers. Wound Repair Regen 2009;17:832e9.
www.elsevier.com/locate/jtv
Clinical study
Treatment of non-healing leg ulcers with honeydew honey Alexander Mayer a,1, Viliam Slezak b,1, Peter Takac c,d, Juraj Olejnik a, Juraj Majtan c,e,* a
Department of Surgery, University Hospital and Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia b University Hospital of the Merciful Brothers, Bratislava, Slovakia c Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia d Scientica, s.r.o., Bratislava, Slovakia e Department of Microbiology, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia
KEYWORDS Honey; Wound; Treatment
Abstract Background: Honey is used as a traditional medicine for centuries by different cultures for the treatment of various disorders. However, not all honeys exhibit equal antimicrobial potency and only a few meet the criteria for clinical usage. Aim: The aim of the study was to determine clinical efficacy of sterilised honeydew honey in the treatment of the lower leg ulcers in 25 patients. Furthermore, we evaluated honey acceptability of patients in terms of pain and overall satisfaction. Methodology: A total of 25 patients with chronic venous leg ulcers were recruited into this study. The 100% g-irradiated sterile honeydew honey was applied onto the cleaned wounds and each wound was assessed at the least two times in for a period of 6 weeks. Results: During the course of treatment, the average wound area of all patients decreased significantly from 51 (3e150) to 22 (0e91) cm2. Eighteen patients (72%) experienced a decrease in reported pain levels while five patients (20%) experienced the same level of pain throughout the study. The overall satisfaction with honey treatment was positive in 80% of patients. Only two patients experienced poor tolerance due to problems at ulcer site related to pain. Conclusion: Based on these findings, honeydew honey has the potential to be one of the medical-grade honeys. ª 2014 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Institute of Zoology, Slovak Academy of Sciences, Dubravska cesta 9, 845 06 Bratislava, Slovakia. Tel.: þ421 2 59302647; fax: þ421 2 59302646. E-mail address: [email protected] (J. Majtan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jtv.2014.08.001 0965-206X/ª 2014 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.
Treatment of non-healing leg ulcers
Introduction Hard-to-heal leg ulcers, or ulcers of a duration longer than 4-weeks and those, which fail to respond to standard care, are the major cause of morbidity in elderly population. Venous or stasis ulcers account for 80%e85% of all leg ulcers [1]. The standard treatment of venous ulcers is the compression of the lower leg by tight bandaging [2] but other treatment options have been proposed including honey therapy. Honey has been used as a traditional medicine for centuries by different cultures for the treatment of various disorders. It offers broad-spectrum antimicrobial properties and promotes rapid wound healing [3]. The antibacterial potential of honey has been considered the exclusive criterion for its wound healing properties. Therefore, antibacterial activity of honey from different floral sources has been intensively studied over the past few decades. Manuka honey, a type of medical-grade honey is the most frequently studied in human clinical trials and claimed to have therapeutic advantages over other honeys. However, some concerns regarding the toxicity of methylglyoxal, and, major antibacterial compound of manuka honey, have been recently expressed [4]. Slovak fir honeydew honey has recently showed excellent antibacterial activity against multi-drug resistant clinical isolates of Stenotrophomonas maltophilia [5] and wound pathogens [6]. It possesses anti-biofilm [6] and anti-MMP-9-activity [7] in vitro. In addition, it has been successfully applied in the treatment of infected gluteofemoral fistulas [8] and as prophylactic agent of endophthalmitis [9]. In this study, we characterised a clinical efficacy of sterilised honeydew honey in the treatment of the lower leg ulcers in 25 patients. Furthermore, we evaluated honey acceptability to patients in terms of pain and overall patient satisfaction.
Patients and methodology A total of 25 patients were recruited for this study. The study protocol was approved by University Hospital ethical committee, and informed consent was obtained from all participants. All patients had chronic venous leg ulcers for more than 12weeks’ duration at the time of presentation, and an ulcer area range of 3e150 cm2. Venous leg ulcers were selected after clinical examination. The baseline demographics of the recruited patients are presented in Table 1. Before the treatment,
95 Table 1
Patient characteristics.
Age (years) Gender Ulcer aetiology Ulcer size (cm2)
Mean Range Male Female Arterial-Venous Mixed Mean Range
79 59e93 12 13 19 (76%) 6 (24%) 51 3e150
ulcers were cleaned with cleansing agents or desloughing materials. The 100% g-irradiated sterile honeydew honey was applied onto the cleaned wounds using sterile spatula, covered by a non-adhering dressing and changed after 1e3-days depending on a wound exudation. Each wound was assessed at the least three times for a period of 3e6 weeks. Primary outcomes such as change in ulcer area, rate of ephithelisation and adverse effects were determined. In addition, all patients were asked to record their subjective feelings (for example, pain level) and overall satisfaction during the honey treatment. Level of pain experienced was assessed as follows: 0 ¼ pain decreased; 1 ¼ pain remained the same; 2 ¼ pain increased. Patients rated the overall satisfaction with the honey treatment on a numerical scale 1e3 (1 ¼ poor, 2 ¼ average, 3 ¼ good).
Results From the twenty-five patients with venous insufficiency or mixed leg ulcers recruited two patients did not complete the study due to problems at ulcer site related to pain. Eighteen patients (72%) experienced a decrease in reported pain levels while five patients (20%) experienced the same level of pain throughout the study (Fig. 1). During the course of treatment, the average wound area of all patients decreased significantly from 51 (3e150) to 22 (0e91) cm2. Honey-induced process of wound healing was also photodocumented in some cases (Fig. 2). Overall tolerance of honey was good after 6 weeks of treatment. In 80% of all assessments, the results were positive; in 12%, there was no change in tolerance; and in 8% there were complaints of poor tolerance (patients dropped out of the study) (Fig. 3).
Discussion In this study, Slovak honeydew honey improved leg ulcer healing within 6 weeks. All patients who
96
Fig. 1 Patients (n ¼ 25) pain evaluation during honey treatment. Level of pain experienced was assessed as follows: 0 ¼ pain decreased; 1 ¼ pain remained the same; 2 ¼ pain increased.
completed study reported overall satisfaction with the honey therapy. Although honey is generally well tolerated, 8% of the patients did not complete the study due to continuous pain. Pain is only known side-effect of using honey (This appears to be due to the acidity of honey, as pain is not experienced when neutralised honey is used): 28% of patients in the largest case series reported pain associated with honey [10]. On the other hand, many authors also documented that most of the patients (60%) experienced a decrease in reported pain levels. This is in accordance with the results of our study where pain was decreased in 72% of the patients. According to a very recent analysis of 25 clinical trials (with a total of 2987 participants) that evaluated the effect of honey on the rate of healing acute wounds (such as burns, lacerations) and chronic wounds (such as skin ulcers, infected
A. Mayer et al. surgical wounds) found that honey dressings do not increase the rates of healing significantly in venous leg ulcers when used as an adjuvant to compression [11]. Nevertheless there are clinical benefits associated with the use of honey in the management of wounds in clinical practice such as reduction in healing duration and treatment costs (consumables and nursing time) and improvement in the quality of life. In addition, authors of above mentioned Cochrane review analysed only studies that report complete wound healing as a primary outcome and did not take into account several clinical studies where wound healing was not achieved but wound size decreased [12]. Limited number of honeys of different botanical and geographical origin is being used clinically. Several other types of honey such as Malaysian tualang honey or Slovak honeydew honey have the potentials being another medical-grade honeys. Honeydew honey may heal wounds through a combination of pathways. It has been shown that honeydew honeys generates higher amounts of hydrogen peroxide than other honeys including manuka honey [13]. Hydrogen peroxide is one of the major antibacterial components and is produced by glucose oxidase e mediated conversion of glucose to gluconic acid under aerobic conditions in diluted honey [14]. Based on several findings [15,16] the overall antibacterial activity of honeys was greatly correlated with H2O2 production in the honeys. Besides the well known honey effects such as osmosis and lowering wound pH, honeydew honey also exhibits a strong antiinflammatory effects partially via inhibition of MMP-9. Recently, it has been postulated that increased levels of pro-inflammatory cytokines as well as the dysregulation of MMP-9 are involved in impaired healing in patients with chronic venous leg ulcers [17]. In particular, the level of MMP-9 remains permanently high and MMP-9 appears to be a major protease responsible for the degradation of matrix and cell growth-promoting agents in chronic wound fluids [18].
Fig. 2 Treatment of leg ulcers with honeydew honey before and after 6 weeks. Honey was sterilized and applied to a depth of 3 mm (20 g of honey to a 10 10 cm area).
Treatment of non-healing leg ulcers
97
References
Fig. 3 The overall patients satisfaction with honey treatment.
According to our very recent study [7], honeydew honey attenuated TNF-a-induced MMP-9 expression in human keratinocytes and suggest that the specific flavonoid content of honey is able to down-regulate the expression of MMP-9. Therefore, the inhibition of MMP-9 secretion depends on the particular flavonoid content of each honey including medical-grade ones.
Conclusions Honeydew honey is a promising wound-healing agent, represents an ideal inexpensive agent that meets all criteria to be therapeutically useful in treating chronic wounds. Thus, honeydew honey has the potential to be one of the medical-grade honeys.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0115-11, and the Operational Program of Research and Development and co-financed with the European Fund for Regional Development (EFRD). Grant: ITMS 26240220020 e “Establishment of biotherapeutic facility and technology proposal for production and development of biodrugs”.
[1] Collins L, Seraj S. Diagnosis and treatment of venous ulcers. Am Fam Physician 2010;81:989e96. [2] Robson MC, Cooper DM, Aslam R, Gould LJ, Harding KG, Margolis DJ, et al. Guidelines for the treatment of venous ulcers. Wound Repair Regen 2006;14:649e62. [3] Molan PC. The evidence supporting the use of honey as a wound dressing. Int J Low Extrem Wounds 2006;5: 40e54. [4] Majtan J. Methylglyoxal-a potential risk factor of manuka honey in healing of diabetic ulcers. Evid Based Complement Alternat Med 2011;2011:295494. [5] Majtan J, Majtanova L, Bohova J, Majtan V. Honeydew honey as a potent antibacterial agent in eradication of multi-drug resistant Stenotrophomonas maltophilia isolates from cancer patients. Phytother Res 2011;25:584e7. [6] Majtan J, Bohova J, Horniackova M, Majtan V. Anti-biofilm activity of natural honey against wound bacteria. In: Majtan J, editor. Honey: current research and clinical applications, vol. 1. New York: Nova Science Publishers, Inc.; 2012. p. 83e106. [7] Majtan J, Bohova J, Garcia-Villalba R, Tomas-Barberan FA, Madakova Z, Majtan T, et al. Fir honeydew honey flavonoids inhibit TNF-a-induced MMP-9 expression in human keratinocytes: a new action of honey in wound healing. Arch Dermatol Res 2013;305:619e27. [8] Vlcekova P, Krutakova B, Takac P, Kozanek M, Salus J, Majtan J. Alternative treatment of gluteofemoral fistulas using honey: a case report. Int Wound J 2012;9:100e3. [9] Cernak M, Majtanova N, Cernak A, Majtan J. Honey prophylaxis reduces the risk of endophthalmitis during perioperative period of eye surgery. Phytother Res 2012; 26:613e6. [10] Dunford CE, Hanano R. Acceptability to patients of a honey dressing for non-healing venous leg ulcers. J Wound Care 2004;13:193e7. [11] Jull AB, Walker N, Deshpande S. Honey as a topical treatment for wounds. Cohrane Database Syst Rev 2013;2: CD005083. [12] Cooper R. Impact of honey as a topical treatment for wounds remains unclear. Evidence-based Med 2014;19:11. [13] Bucekova M, Valachova I, Kohutova L, Prochazka E, Klaudiny J, Majtan J. Honeybee glucose oxidase e its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys. Naturwissenschaften 2014;101:661e70. [14] White JW, Subers MH, Schepartz AI. The identification of inhibine, the antibacterial factor in honey, as hydrogenperoxide and its origin in a honey glucose-oxidase system. Biochim Biophys Acta 1963;73:57e70. [15] Brudzynski K. Effect of hydrogen peroxide on antibacterial activities of Canadian honeys. Can J Microbiol 2006;52: 1228e37. [16] Chen C, Campbell LT, Blair SE, Carter DA. The effect of standard heat and filtration processing procedures on antimicrobial activity and hydrogen peroxide levels in honey. Front Microbiol 2012;3:1e8. [17] Serra R, Buffone G, Falcone D, Molinari V, Scaramuzzino M, Gallelli L, et al. Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin. Wound Repair Regen 2013; 21:395e401. [18] Moor AN, Vachon DJ, Gould LJ. Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers. Wound Repair Regen 2009;17:832e9.