manuka honey

Survey of the antimicrobial activity

of Australian honey

Julie Irish

Dee Carter

Shona BlairSchool of Molecular and Microbial Biosciences

University of Sydney

Antimicrobial activity of honey

Honey has been well studied for its antimicrobial and wound healing properties

Low water activity ~80% sugars

Low pH Gluconic acid pH 3.2 – 4.5

Hydrogen peroxide Glucose oxidase

Antimicrobial activity of honey

Leptospermum honey Leptospermum honey + catalase

Brush box honey Brush box honey + catalase

Staphylococcus aureus

Floral factors Non-peroxide activity (Leptospermum spp.) Also known as Unique Manuka Factor (UMF®) Advantage in clinical situations

Non-peroxide activity is caused by methylglyoxal (MGO) High concentrations of MGO in manuka

honey that correlate strongly with non-peroxide activity

MGO is naturally produced in all cells as a by-product of metabolism

Highly toxic Modifies DNA, RNA, and proteins Targets them for degradation by the cell’s own

machinery Some bacteria and fungi have well-defined

detoxification systems

How does this fit with the current theory about medicinal honey? MGO produced by stressed plants May explain why some organisms are more sensitive

to honey than others Combination of MGO and honey makes it non-toxic to

human cells?

Need further studies on source, mode of action, standardisation in medicinal honeys

Non-peroxide activity is caused by methylglyoxal (MGO)

Survey of Australian honeys for antibacterial activity

Important to find the most highly active honeys for therapeutic use

345 New Zealand honeys tested (Allen et al. (1991))

No published data for Australian honey

Overview of activity of Australian honey

Testing the antibacterial activity of honey Compares the antibacterial activity of a given honey to

that of phenol Staphylococcus aureus is mixed into agar plate Holes cut into agar Various solutions added to wells

25% honey in water

(tests total activity)

OR 25% honey in catalase solution

(tests non-peroxide activity)

OR Solutions of 2 to 7% phenol

(standard curve)

Solutions diffuse out of the wells during incubation

If they prevent the growth of S. aureus

zones of inhibition Zones are measured,

mean diameter squared Standard curve generated

from phenol solutions

Testing the antibacterial activity of honey

y = 137.86x - 108.1

R2 = 0.9889

0 1 2 3 4 5 6 7 8

Phenol concenration (% w/v)

Testing the antibacterial activity of honey

Honey compared to phenol (x 4.69 for dilution and density)

400 mm2 zone of inhibition = 17.3% phenol equivalent

The higher the number, the more active the honey

Allows comparison between different honeys

y = 137.86x - 108.1

R2 = 0.9889

0 1 2 3 4 5 6 7 8

Phenol concenration (% w/v)

Results

503 honey samples tested 477 Apis mellifera 26 Trigona spp.

Activity (phenol equivalent) varies greatly <5 Insignificant therapeutic value 5-10 Low activity >10 Therapeutically beneficial >20 Highly active

Total activity<5 5 – 10 10 – 20 >20 Sum

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

(% of country)

Sum >10(% of state)

NSW 93 7 135 40 275 (57.7%) 175 (64%)

VIC 14 1 2 4 21 (4.4%) 6 (29%)

TAS 23 2 20 1 46 (9.6%) 21 (46%)

QLD 12 0 7 9 28 (5.9%) 16 (57%)

SA 10 0 5 0 15 (3.2%) 5 (33%)

WA 39 2 20 31 92 (19.2%) 51 (55%)

Sum 191

(2.5%)

(39.6%)

(17.9%)

(100%)

(57.5%)

60% of all samples had antibacterial activity

57.5% of all samples had therapeutically beneficial antibacterial activity

Distribution of total activity

020406080

100120140160180200

<5 5-10 10-15 15-20 20-25 25-30 30-35

Total antibacterial activity (% (w/v) phenol equivalent)

Of the honeys that have antibacterial activity, most are in the therapeutically beneficial range

Average total activity (n=286): 17.8 5 (7.4 – 34.3)

Non-peroxide activity<5 5 – 10 10 – 20 >20 Sum

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

(% of country)

Sum >10(% of state)

NSW 209 6 50 10 275 (57.7%) 60 (22%)

VIC 21 0 0 0 21 (4.4%) 0 (0%)

TAS 43 0 3 0 46 (9.6%) 3 (6.5%)

QLD 22 1 1 4 28 (5.9%) 5 (18%)

SA 14 1 0 0 15 (3.2%) 0 (0%)

WA 88 4 0 0 92 (19.2%) 0 (0%)

Sum 397

(2.6%)

(11.4%)

(3.0%)

(100%)

(14.3%)

17% of all samples had non-peroxide activity

14.3% of all samples had therapeutically beneficial non-peroxide activity

Distribution of non-peroxide activity

<5 5-10 10-15 15-20 20-25 25-30

Antibacterial activity (% (w/v) phenol equivalent)

Of the honeys that have non-peroxide activity, most are in the therapeutically beneficial range

High non-peroxide activity is rare

5-10 10-15 15-20 20-25 25-30

Antibacterial activity (% (w/v) phenol equivalent)

Non-peroxide activity

Of the 80 honeys with non-peroxide activity, 62 (77.5%) were from Leptospermum, or contained Leptospermum

Mean non-peroxide activity 17.2 4.1 (9.8 – 25.9)

Some species/areas more reliable than others L. polygalifolium (Jelly bush): 29 samples, 28 had non-

peroxide activity L. scoparium (Manuka): 11 samples, none had non-

peroxide activity, some inactive Most active Leptospermum honeys from NSW-QLD border

18 non-Leptospermum samples had non-peroxide activity

Mean non-peroxide activity 10.1 1.7 (8.1 – 15.9)

Spotted gum (3/6 samples) South west Tasmanian wildflowers (3/5 samples) Stonefruit orchard (1/2 samples) Clover (1/5 samples) Melaleuca (2/28 samples) Moort (1/1 samples) Forest red gum (1/2 samples) Messmate (1/6 samples) Unspecified mixed flora (5/64 samples)

Floral sources 477 honey samples from 142 different floral

sources, including mixed flora 78% of honeys were from native

Australian flora, another 17% were likely to contain natives

38% of honeys contained eucalypts

24% of honeys contained Leptospermum (likely to be biased)

Eastern Australia

Inactive

Hydrogen peroxide activity

Eastern Australia

Highest total activity: 34.3 (Apple box/Red stringybark)

Highest non-peroxide activity: 25.9 (Jelly bush)

High non-peroxide activity (>20% phenol equivalent) in Leptospermum samples from QLD and northern NSW

12 non-Leptospermum honeys

had non-peroxide activity

Western Australia

Inactive

Hydrogen peroxide activity

Western Australia Highest total activity: 31.9 (Stonefruit) Highest non-peroxide activity: 9.7 (Melaleuca) 31 honeys with total activity >20

(many from flora endemic to WA)

Jarrah: 19 samples Activity range 17.7 to 31.4 1 sample was inactive

Karri: 3 samples Activity range 18.5 to 29.6

Marri: 9 samples Activity range 18 to 29.7 1 sample was inactive

Important message

It is impossible to predict the activity of a given honey, must test every batch 32 samples, same beekeeper, same floral source,

same time, different hives 31 samples had total activity between 11 and 19 1 sample was inactive

30% of Leptospermum honeys were inactive,

15% had hydrogen peroxide activity only Activity relates to floral source to an extent, but there

are clearly many other factors involved Environmental conditions, soil type, soil and plant

microbiology, bee health…

Stingless bee honey

26 samples of Trigona spp. honey from QLD

Mixed flora, various times of year

23 samples had total activity >20 (12.4 – 32.1)

24 samples had non-peroxide activity >10 (11.5 – 23.7)

Conclusions Australian honeys show a wide range of

antibacterial activities Many have potential for therapeutic use Non-peroxide activity exists in honeys from

various floral sources Leptospermum spp. remain the most

reliable source of high non-peroxide activity Statistical analysis is ongoing Identified areas to focus on in

future studies Role of MGO?

Acknowledgments

The 124 beekeepers who supplied honey samples for the survey

Rob Manning, Dept of Agriculture, WA Tim Heard, CSIRO Entomology, QLD Comvita, New Zealand RIRDC

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