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Contents
Background: Honey and Wound Care ............................................................................... 1
Sourcing and Variability ...................................................................................................... 1
Antioxidant activity .............................................................................................................. 2
Antibacterial Activity ............................................................................................................ 2
Case Studies and Clinical Trials ......................................................................................... 5
Conclusions: ........................................................................................................................ 7
Anti-infective and Anti-inflammatory Activities of Manuka Honey: A
Review of the Current State
Background: Honey and Wound Care
Honey has been used since 2500 BC as a wound dressing, its physical
properties allowing it to serve as a viscous ointment for topical treatments
providing an effective barrier (Lu et al. 2014). But the eventual resolution of
wound healing requires coordination across numerous physiological processes
which benefit from the chemical makeup of honey that provides antibacterial and
antioxidant activities. Recognition of Manuka honey as especially efficacious for
wound treatment began in the mid-1980s when Peter Molan of Waikato
University in New Zealand began reporting on its activity against a wide range of
bacterial strains (Carter et al. 2016). An important indication of the recognition
that Leptospermum based honey has achieved for efficacy, is registration under
the medical authorities of Australia, Canada, the EU, Hong Kong, New Zealand
and the US as a medical device for use in wound dressings (Jenkins and Cooper
2012; Cokcetin et al. 2016). There is considerable need for new and effective
interventions to address chronic wounds; a survey conducted in 2012 of the US
Wound Registry determined that chronic wounds, wounds that have not fully
healed after a period of two months, affect 6.5 million people across the US
incurring enormous costs in health care resources and lost time, pain and
suffering. This trend is only expected to increase as the population ages further
(Fife and Carter 2012). It is critically important then to explore new modalities for
addressing the bacterial infections and inflammation that contribute to the failure
to achieve wound resolution.
Sourcing and Variability
Honey is first, a food source, highly concentrated in the sugars fructose and
glucose, but also including a wide range of additional components that make
essential contributions to its nutritional and medicinal properties. Like all natural,
biological products, its exact make up will vary significantly depending on seasonal
and environmental factors and on the age and health of the bee colony (Ohashi,
Natori, and Kubo 1999). But medicinal activity appears to depend especially on
the raw material of the honey’s manufacture, that is, the floral source of the nectar.
Honey can be categorized as multi- or polyfloral when it has been gathered from
a variety of plant species as will occur in a forest or meadow setting. If wholly
derived from just one species of flower it is termed monofloral. The term "Manuka
honey" indicates honey derived from the Manuka tree, Leptospermum scoparium,
of the family Myrtaceae, that grows as a shrub or small tree throughout New
Zealand and eastern Australia (Kato et al. 2012). The possibility of standardizing
a honey by composition and biological efficacy increases markedly with a
monofloral honey (Alvarez-Suarez et al. 2014).
Antioxidant activity
The yellow-gold color of honey is imparted by flavonoids, those predominant in
Manuka honey are pinobanksin, pinocembrin and chrysin. In addition to attracting
pollinators, flavonoids are also effective antioxidants. An important aspect of the
wound healing activity is honey's ability to quench reactive oxygen species (ROS)
reducing further cell necrosis and tissue damage and helping to prevent fibrosis
during healing (Molan 2001). While the levels of polyphenolics present in Manuka
honey would be expected to contribute to the antioxidant capacity, its scavenging
capacity against free radicals is further supported by methyl syringate (MSYR) and
its glycoside, methyl syringate 4-O-beta-D-gentiobiose (known commonly as
leptosin) which together serve as inhibitors of myeloperoxidase. Leptosin is unique
to Oceanic honeys being particularly abundant in Manuka and has been proposed
as a potentially useful chemical marker for purity. Levels of leptosin show a
positive correlation with antimicrobial activity (Kato et al. 2012).
Antibacterial Activity
The search for the basis of the antibacterial properties of honey was a subject of
Victorian science initiated by van Ketel in 1892 (Dustmann 1979). The
bactericidal/bacteriostatic activity common to various honeys is provided by
biochemical and physicochemical factors including high osmotic pressure and low
pH. The osmotic gradient created by the elevated levels of solutes contributes to
desiccation of microorganisms and acidity slows growth.
Some of the same physical/chemical properties which inhibit microbial growth also
promote tissue regeneration. The osmotic gradient draws fluid from circulation into
the wound. The resulting layer of diluted honey that is continually created helps to
prevent adherence of dressings to fragile healing tissues avoiding tearing and
helping to reduce the need for surgical debridement (Subrahmanyam 1993; Efem
1993). Acidification of the wound bed by honey's low pH promotes the release of
oxygen from hemoglobin helping to alleviate the low oxygen tension under
dressings (Kaufman et al. 1985). Another potential aid to healing is the observation
that honey stimulates the growth both of epithelial cells and fibroblasts speeding
the closing of wounds without the need for skin grafts (Subrahmanyam 1998; Efem
1993).
However, even if the honey is diluted to reduce osmolarity to negligible levels and
buffered to raise the pH to neutrality, antimicrobial activity is still observed.
Although the same phenolic compounds providing the antioxidant activity in honey
are known to also possess antibacterial activity, the concentrations present in
honey are insufficient to significantly inhibit bacterial growth (Mavric et al. 2008).
In many honeys the antimicrobial activity is provided by hydrogen peroxide (Carter
et al. 2016). The H2O2 is derived from glucose oxidase, secreted from the bees,
reacting with glucose, but peroxide levels are low in Manuka relative to other
honeys. The antibacterial activity that remains following the elimination of
incidental H2O2 is termed non-peroxide activity (NPA) which is equivalent to the
trademarked designation Unique Manuka Factor (UMF) registered by the UMF™
Honey Association and available under license for Manuka producers in New
Zealand (Cokcetin et al. 2016). The UMF is particularly valuable because unlike
hydrogen peroxide it is not destroyed by endogenous human catalase activity and
while it is heat labile and cannot be autoclaved, it will withstand sterilization,
required for wound dressings, by gamma irradiation used to kill clostridial spores
which can occur in honey (Molan and Allen 1996).
The antibacterial activity used to grade the UMF value is typically determined by
an agar well diffusion assay of S. aureus. A reference curve is generated by
assaying titrations of phenol. A UMF equivalent in activity to 10% phenol is
generally considered therapeutically active (Cokcetin et al. 2016). Electron
microscopic analysis of Manuka treated S. aureus cultures revealed cell
morphology suggesting that cell division was disrupted by failure to complete the
cycle, leading to an accumulation of cells with finished septa failing to separate,
but which were otherwise normal in appearance suggesting a bacteriostatic
mechanism (Henriques et al. 2010).
The factor most commonly cited as the source of NPA in Manuka honey is a simple
dicarbonyl compound: methylglyoxal (MGO). MGO is found in other food sources,
for example it is formed during coffee roasting in amounts of 23–47 ppm (Hayashi
and Shibamoto 1985). Dihydroxy acetone (DHA) is derived from Manuka nectar
and as honey ripens, it is converted to MGO (Mavric et al. 2008; Adams, Manley-
Harris, and Molan 2009). In New Zealand Manuka honey MGO levels have been
shown to be closely correlated to NPA across MGO concentrations from 200 - 800
mg/kg. MGO was unambiguously identified by Mavric et al in 2008 in various New
Zealand Manuka honey samples when the structure was confirmed by LC-TOF-
Mass Spectrometry. A survey of the antibacterial activities of honeys from various
sources showed that only the Manuka honeys were able to significantly inhibit
bacterial growth at concentrations from 15-30% (w/v) whereas honey from other
sources failed at concentrations lower than 80%. It was also noted that the
antibacterial activities of the Manuka samples were directly related to the
concentrations of MGO in the honey. Various dicarbonyl compounds displayed
antibacterial activity against E. coli and S. aureus., but the potency of MGO was
several-fold greater than glyoxal or 3-deoxyglucosulose showing a minimally
inhibitory concentration of 1.1 mM. The dilutions of Manuka, 15-30%, that
possessed antibacterial activity were determined to contain MGO at
concentrations of 1.1 to 1.8 mM; sufficient to account for the observed activity.
Finally, when a 20% solution of otherwise inactive honey was spiked with neat
MGO to a final concentration of 1.9 mM, inhibition of bacterial growth equivalent to
native Manuka honey was observed (Mavric et al. 2008). It can be predicted then
that a honey with MGO concentrations greater than 260 mg/kg can be expected to
have antibacterial activity meeting the therapeutic threshold of >10% phenol
equivalents (Adams et al. 2008; Atrott and Henle 2009).
A survey of Australian honeys derived from various species of Leptosporum
showed that under the right storage conditions (in the dark under refrigeration at
4C) the NPA is remarkably stable showing little change over a period of seven
years (Cokcetin et al. 2016). Samples were derivatized with PFBHA reagent and
quantified by Reverse Phase-High Performance Liquid Chromatography (RP-
HPLC) as described by Windsor (Sarah Windsor 2012). Prior to conducting dose
response experiments, honey samples were treated with catalase to remove any
potential H2O2 activity, then assessed for their ability to inhibit the growth of S.
aureus ATCC 25923. An exceptionally strong correlation (r2 = 0.95, Pearson's rho
= 0.97, p<0.05) was established between the antibacterial activity and MGO
concentrations. The highest levels of activity (>20% phenol equivalence from >800
mg/kg MGO) were associated with monofloral samples. When grouped by source
species, the honey samples did not show a signficant reduction in mean antibiotic
activity following the seven years of storage. Only two samples lost activity
completely and 23 of the L. polygalifolium samples showed increases in NPA
possibly due to further conversion of DHA to MGO (Cokcetin et al. 2016).
Concentrations of MGO measured in Manuka are up to 100-fold greater than that
found in conventional honeys (Mavric et al. 2008). But not all honeys derived from
Leptospermum sources show antibacterial activity and even when restricted to the
species L. scoparium and L. polygalifolium, the MGO levels can vary by more than
an order of magnitude (Sarah Windsor 2012). Another important chemical marker,
also quantifiable by HPLC, is hydroxymethylfurfural (HMF). It can be found in all
types of honey and serves as an indicator of spoilage; if levels exceed 40 mg/kg
the product cannot be exported or sold. No relation between HMF and MGO levels
has been observed (Cokcetin et al. 2016).
The formation of biofilms is especially problematic in bacterial infections of wounds
and in oral care. The films are a gel-like matrix of polysaccharides and other
components in which pathogens embed. The biofilm matrix limits the access of
antibiotics to the pathogens to the extent that the dose effectiveness can be
reduced by as much as three orders of magnitude (Hoyle and Costerton 1991).
The continuing release of pathogenic cells from the matrix can contribute to
sustaining chronic inflammation in the wound (Alvarez-Suarez et al. 2014; Ngo,
Vickery, and Deva 2012). Biofilms are common to chronic wounds contributing to
the difficulty in achieving satisfactory healing (Woodward 2019).
Four honeys were compared for their ability both to prevent the initial formation of
biofilms and degrade biofilms that had already been established. The most
effective honey, both in terms of preventing and degrading S. aureus biofilms was
a monofloral (Leptospermum scoparium var. incanum) Manuka honey with 958
ppm MGO that caused a 95% reduction in biofilm mass accumulation, at a
concentration of 8% (w\v) in vitro. Slightly less active was a commercial medical
grade Manuka/Kanuka combination, Medihoney®, with 776 ppm MGO.
Interestingly MGO, at concentrations equivalent to those present in the Manuka
honeys and in combination with an artificial solution of sugars found in honey (45%
glucose, 48% fructose, 1% sucrose) was also able to inhibit formation of biofilms,
but was far less potent than the complete, natural honeys. Further, the MGO-
sugar solutions were wholly ineffective against established biofilms. This suggests
either that other components in complete and natural Manuka honey besides MGO
contribute significantly to the efficacy against biofilms or that MGO in the context
of complete honey is better able to access and act on the embedded pathogens
(Lu et al. 2014).
Another antibacterial factor not related to hydrogen peroxide is the amphipathic
protein, bee defensin-1 (previously royalisin). The mature protein contains 51
residues (5.5 kDa) with potent activity against gram negative, but not gram positive
bacteria (Fujiwara et al. 1990). This protein was readily detected in samples of the
medical grade honey Revamil® which is produced under controlled greenhouse
conditions in The Netherlands. Following electrophoretic separation of Revamil
sourced (RS) samples on native polyacrylamide gels, the location of defensin-1
can be visualized by overlay bioassays with B. subtilis, similar analyses of a
Manuka honey with UMFTM of 16+ yielded no defensin-1 related response. The
two honey samples also differed in hydrogen peroxide levels: RS samples diluted
to 40% (v/v) accumulated 3.5 mM H2O2 after 24 h, Manuka treated in the same
manner showed no detectable H2O2. In contrast the Manuka samples contained
over 40-fold higher concentrations of MGO than the RS samples. The bactericidal
activity of the two honeys was compared directly by conducting dose response
assays against a battery of strains including Bacillus subtilis ATCC6633,
Escherichia coli ML-35p, Pseudomonas aeruginosa PAO-1 (ATCC 15692) and
against methicillin-resistant Staphylococcus aureus (MRSA) strain AMC201. The
RS honey displayed a more rapid bactericidal activity showing greater potency
when assessed after a two-hour exposure; however, after 24 h the Manuka sample
proved to be significantly more inhibitory than the RS sample showing 8-fold
greater activity against MRSA. Against B. subtilis and E. coli Manuka was twice
as potent while the activity of both the Manuka and RS samples were equivalent
on P. aeruginosa after 24 h. The multivalent nature of the Manuka sample was
demonstrated in follow-up assays where the MGO in the honey was neutralized by
chemical conversion to the non-bactericidal S-lactoylglutathione. This step
eliminated the activity against MRSA, but the activity against E. coli was unaffected
and though toxicity towards B. subtilis and P. aeruginosa was significantly reduced,
it was not eliminated (Kwakman et al. 2011).
The variety of antiinfective agents identified in honey points to an important
aspect of its utility for future use, this diversity of activities greatly reduces the
potential for bacterial strains to develop resistance relative to pharmaceuticals
that target a single metabolic locus. There have not yet been any reports of
clinical instances of acquired resistance to honey. Attempts have been made to
generate such strains from S. aureus, S. epidermidis, E. coli and P. aeruginosa,
in a laboratory setting, by culturing under sub-lethal concentrations, but honey
resistant mutants were not generated (Cooper et al. 2010; Blair et al. 2009). A
practical demonstration of this aspect of honey's antibacterial activity is given in
the results of a screen conducted against MRSA and P. aeruginosa. Sublethal
concentrations of Manuka honey were tested in combination with fifteen
antibiotics, yielding five combination therapies of honey and antibiotics that
significantly improved in vitro efficacy against these two important wound
pathogens (Jenkins and Cooper 2012). An especially attractive combined
treatment might be the combination of honey as a topical dressing used with a
systemic antibiotic that could attack circulating pathogens (Carter et al. 2016).
Synergistic activity observed in vitro by combinations of Manuka and antibiotics
against a MRSA strain have included oxacillin, tetracycline, imipenem and
mupirocin (Jenkins and Cooper 2012).
Case Studies and Clinical Trials
Honey as a dressing was compared directly to silver sulfadiazine in a trial with 52
patients in each treatment group. Under the honey treatments healthy granulation
tissue appeared in almost half the time required for the silver sulfadiazine
treatments. The great majority of honey patients (91%) achieved sterility within a
week where only a few of those on the silver sulfadiazine treatment (7%) managed
control in the same time period. After two weeks, 87% of the honey treated wounds
had healed whereas only 10% of the silver sulfadiazine dressed wounds were
assessed as healed. The frequency of hypertrophic scarring and postburn
contracture was also lower in the honey treatment group (Subrahmanyam 1991).
An evaluation of honey impregnated gauze vs a polyurethane film (OpSite®) was
conducted in a randomized trial of 46 patients per treatment group. The results
showed that used as a cover for fresh partial thickness burns, honey treated
wounds healed significantly faster requiring on average only about two-thirds the
time needed for healing under the polyurethane film and reducing the frequency of
infection by over 50% relative to the film treatment (Subrahmanyam 1993). In a
second trial again evaluating treatment of partial thickness burns, a similar honey
impregnated gauze dressing was compared to dressings made with amniotic
membrane, obtained from separations of the chorion and placenta shorty following
delivery. Patients varied widely in age from 3 to 62 years of age. Burn injuries
treated with honey required about half as many days on average for healing as
compared to the membrane dressed patients (P < 0.001). In the honey treatment
group of those who tested initially as positive for bacterial infection, 82% achieved
sterility after one week whereas 58% of the patients who had presented with
infections were sterile by the same time under the membrane dressing
(Subrahmanyam 1994).
In a prospective trial of 15 patients with abdominal wound disruptions following
Cesarean sections, honey was applied rather than conventional dressing.
Excellent results were obtained for all cases after two weeks. Comparing their
responses retrospectively to 19 patients who had been treated with hydrogen
peroxide, hypochlorite solution and saline soaked gauze, the honey treatment
group benefitted in requiring on average, only half as many days of hospitalization.
The honey treatment combined with the use of micropore tape for approximation
avoided the need for general anesthesia and resuturing (Phuapradit and Saropala
1992).
The overhydration of skin, can initiate inflammation as the penetration of irritants
through the dermis can be increased. In the specific case of overhydration due to
incontinence this can give rise to incontinence-associated dermatitis (IAD). The
overly hydrated, inflamed skin then also becomes more susceptible to tears and
mechanical injury. If the condition becomes chronic, superations can further
reduce skin integrity as exudates contain proteases that weaken the dermal matrix.
This is especially a risk in older subjects with reduced epidermal thickness
(Woodward 2019).
Medihoney® Barrier Cream (MBC) is a commercial product, silicone based,
including 30% (w/v) Medihoney® Antibacterial Honey. A pilot scale, singly blinded,
multicenter trial examined MBC as an intervention for intertrigo (moisture
associated damage in large skin folds). The subjects randomized to this study
presented with bilateral intertrigo and so served as their own study controls.
Subjects reported a statistically significant reduction in pain and less than half as
many pruritis complaints with the MBC treatment as compared to a zinc oxide
ointment although no significant differences in the course of wound healing was
observed (Nijhuis et al. 2012).
The high degree of safety associated with MBC was demonstrated in a recent case
study where a 2 month old infant who had developed a persistent rash that resisted
treatment at home and in the hospital with several skin treatments and protectant
gels. Applications of MBC finally initiated significant improvements showing
epithelialization after one week and a cessation of apparent pain and discomfort
(Woodward 2019).
Mupirocin (Bactroban®) is often used for treatment of nasal colonization by
methicillin resistant S. aureus, but the appearance of resistance limits continuous
use. In a trial evaluating Manuka honey for nasal localized MRSA, patients older
than 18 years received medical grade Manuka honey or 2% mupirocin 3 times per
day over a 5 day period. Although the incidence of decolonization achieved by the
drug (57%) was numerically greater than that observed for honey (43%) the
difference between the two treatments was not statistically significant. Importantly,
the rate of acquisition of new resistance to mupirocin during the trial was almost
10% and the study sponsors concluded that the honey provided a potential
strategy for achieving significant treatment while addressing resistance
(Poovelikunnel et al. 2018).
Atopic dermatitis (AD) is characterized by pruritus and skin rash, it is more common
in childhood and can affect as many as a fifth of children, but can persist into 1-3%
of adults (Schneider et al. 2013). In a small, open-label, pilot-scale trial, 14 adult
(mean age 33) sufferers with AD affecting bilaterally similar areas were issued
Medihoney® and sterile gauze. At baseline, the affected areas were graded by
the Three Item Severity score (TIS), which accounts for erythema,
edema/papulation, and excoriation (Wolkerstorfer et al. 1999). For seven days,
the subjects applied honey and gauze to the treatment site in the evening, and
removed the dressing and washed the site each morning. The other lateral site
was left untreated and served as the control. At the end of the week of treatment,
both sites were again scored on the TIS scale. The severity of the dermatitis, was
significantly reduced by the honey treatment as assessed by the TIS score
(p<0.001). This trial was then followed up with in vitro experiments conducted in
an attempt to identify a possible mechanism of action for the activity that was
observed. The inflammatory chemokine CCL26 released by keratinocytes plays
an important role in the development of AD (Owczarek et al. 2010). The ability of
cultured HaCaT cells, a model of keratinocytes, to secrete CCL26 following IL4
induction, was significantly inhibited by in vitro honey treatments to ~50% of
maximum signal in the presence of 1% w/v honey. The itching and edema
associated with AD is further exacerbated by the release of histamine by Mast cells,
in vitro honey treatment also showed significant inhibition of histamine release from
cultured LAD2 cells. As swabs which had been taken from the subjects in the AD
trial had indicated that the Medihoney® treatments had not changed the skin
bacterial flora, these results suggest that the relative relief that had been achieved
was due to anti-inflammatory effects by the honey rather than anti-bacterial activity
(Alangari et al. 2017).
A compelling indication of the wide safety margins that Manuka honey provides for
use on delicate tissues is its application in ophthalmic treatments. Corneal edema
is a common complication of cataract surgery leading to a thickening of the cornea
and affecting vision. A retrospective studied reviewed the outcomes of 18 cases
of persistent corneal edema following ocular surgery treated with Optimel™
Antibacterial Manuka Eye Drops 2 to 3 times daily. With continual use reductions
in corneal thickness and improvements in visual acuity were achieved. No
significant adverse events were reported (Albietz and Lenton 2015).
Manuka honey has also been assessed for potential protection against tooth decay.
In in vitro experiments evaluating antibacterial activity against cariogenic S.
mutans and Lactobacillus strains, a 25% w/v solution of Manuka honey was
significantly inhibitory towards both strains and was statistically significantly more
active than equal concentrations of Dabur (Indian) honey (Beena et al., n.d.).
Recently, a relatively large (n=135), double-blinded, randomized controlled trial
was conducted with 12 - 15 year-old school children evaluating mouth washes for
both gingival and plaque control. Dental plaque is an example of a biofilm
supporting multiple strain of bacteria. One of three mouthwash treatments, based
either on Manuka honey, a raw honey or 0.2% chlorhexidine (CHX), was
administered twice daily for 3 weeks. Gingival and plaque indices were scored at
baseline, and one day and one week after the end of the trial. Both honeys caused
statistically significant reductions in scoring for plaque of greater than 50% while
CHX treatments scored reductions of two-thirds. CHX also reduced the score for
gingivitis by about two-thirds whereas the honey mouthwashes led to a reduction
of about 35% (Singhal et al. 2018). There were no adverse events reported among
the child patients and no staining of subject’s teeth by the honey mouthwashes
which is commonly observed with CHX (Jones 1997).
Conclusions:
A considerable body of evidence has accumulated, both mechanistic and clinical
to elevate consideration of Manuka honey from a folk remedy to an intervention for
a variety of conditions. The advantages Manuka honey provides to the modern
pharmacopeia is a combination of antibacterial and anti-inflammatory activities
providing promise for problematic chronic conditions while delivering those
activities in a context sufficiently gentle that it can be used in direct applications to
the eye and for use in children. But just as importantly, given concerns about
antibacterial resistance is the evidence from the laboratory and from clinical
experience that interventions with medicinal honey have shown no potential, to
date, for generating microbial resistance. It is hoped that the examples provided
here, far from exhaustive, might give rise to further research in the indications cited
or provide ideas for applications for new, currently unforeseen health benefits.
For a source of research grade Manuka honey with a consistently high UMF factor,
contact NZ-Ruzio Llc at http://www.honeyforhealing.com. Samples can be made
available for supporting studies following a review of protocols.
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