Introduction

Honey has been used for the treatment of infected wounds hundreds of years ago, even before the discovery of bacteria as causes of infections.¹ In 50 A.D, Dioscorides described honey as being effective for all rotten and hollow ulcers. ¹ The bactericidal action of pure honey on many pathogenic organisms including enteropathogens such as Salmonella species, Shigella species, Escherichia coli and other gram negative organisms has also been reported. ² Furthermore, honey has been employed to shorten the duration of diarrhoea in patients with bactericidal gastro-enteritis due to bacterial infection as well as applied to heal wounds like the conventional antibiotics and antiseptics. ^3-5

The current antibiotic – resistant microbial species, for example, Pseudomonas and Klebsiella species resistance to gentamicin, amikacin and ceftazidine, ⁶ as well as toxicity to conventional therapy among other factors, have led to resurgence of ancient remedies.  Honey has been employed by individuals in this environment for its numerous therapeutic benefits, since as a natural product it produces very few adverse effects. ^7-9 Of utmost importance to the authors is its antimicrobial actions.

 The present study was designed to screen for the antimicrobial spectrum and efficacy of a type of honey obtained from OgunState, in Nigeria, using a few selected gram negative and gram positive bacteria and to also compare its effect with standard drugs, in order to justify its replacement of conventional drugs by some indigenes. The physical, chemical and biological properties of honey have already been documented. ^10-13

Materials and Methods

Source and dilution of honey

The honey used in this study was obtained from OgunState in Nigeria. It was diluted in sterile distilled water to different concentrations of 12.5%, 25.0%, 50.0% (v/v). 100.0% honey was referred to as ‘neat’.

Source and dilution of standard drugs

A concentration of 0.2% ciprofloxacin, 2 mg/ml (ampoule) marketed as Cifran (Fidson Drugs Nig. Ltd) was obtained for use. Tetracycline (Tetracap, Fidson Drugs Nig. Ltd) 250 mg was emptied into 10 ml sterile distilled water to make up the stock solution of 25 mg/ml. 10ug/ml was used for gram positive while 50ug/ml was employed for gram negative bacteria.

Organisms

Standard isolates and strains of micro-organisms namely: Escherichia coli, Staphylococcus aureus, Streptococcus faecalis, Staphylococcus albus, Klebsiella sp., Proteus mirabilis, Peudomonas aeruginosa and Candida albicans were employed for both sensitivity tests and the determination of minimum inhibitory concentration (MIC).

In-vitro demonstration of antimicrobial activity (Sensitivity tests)

The method employed ^{14, 15} has been widely used for antimicrobial susceptibility testing. Mueller Hinton and MacConkey agars were prepared according to the manufacturer’s instructions. Pure culture of micro-organisms was grown on nutrient agar. Five colonies of each organism were picked using an inoculation loop into the Mueller Hinton broth (Oxoid, England) incubated for 4 h at 37°C, diluted with sterile saline to a density visually equivalent to 10⁶ cfu/ml, which corresponded to MacFarland standard. The suspension was then seeded evenly onto the surface of Mueller Hinton agar plates (Oxoid, England) in triplicates with a sterile swab.  Using a sterile 6 mm diameter cock borer, 5 wells were cut in the agar to which appropriate concentrations of honey were added, as well as the standard drugs, ciprofloxacin and tetracycline separately, which served as the controls. The plates were incubated at 37°C for 48 h under aerobic condition and were thereafter examined at 24 h for zone of inhibition and again at 48 h. C. albicans was grown on Sabouraud dextrose medium (Oxoid, England) and processed as above.

Minimum inhibitory concentration (Broth dilution method)

Appropriate volumes of honey was added to sterile tubes containing 10.0 ml of Mueller Hinton broth (Oxoid, England) to give a final concentrations of 512mg/ml, 256mg/ml, 128mg/ml, 64mg/ml, 32mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 2mg/ml and 1mg/ml. Using a volumetric pipette, 50µl of the test bacteria and fungal broth cultures were added into each of the tubes. The tubes containing the bacterial cultures were incubated at 37°C for 24 h, while the candida culture was incubated at 37°C for 48 h, after which they were read macroscopically to determine the lowest concentration of the test honey that did not permit any visible growth when compared with that of the control.

Two controls were employed; one was a row of positive control tubes containing only the growth medium and each of the micro-organisms, while the other was a negative control which consisted of a row of tubes containing different concentrations of honey with no organism.  The minimum bactericidal concentration (MBC) was also determined.

Phytochemical analysis of honey

Phytochemical tests ¹⁶ were carried out on pure honey. A few millimeter sample of honey was mixed with aqueous sulphuric acid and benzene to test for presence of anthraquinone glycosides.  Also, Mayer and Dragendorff’s reagents were employed to test for presence of alkaloids. With the addition of gelatin and ferric chloride solution, test for tannins was conducted. Keller-keliani and Legal tests were carried out for cardiac glycosides, froth and haemolysis tests for saponins and 1%aluminium chloride solution in methanol was used to investigate flavonoids. Lastly, Benedict’s and Fehling’s solutions were added to determine presence of reducing sugars.

Results

The data obtained showed inhibitory effects of honey at 50 and 100% concentrations on the various investigated microorganisms as being dose-dependent (Table 1). However, both 50% and 100% honey did not produce any inhibition of S. faecalis.

In all cases of micro-organisms tested, the neat concentration of honey produced a greater inhibition than tetracycline except for E. coli, whereas, ciprofloxacin at the employed concentration of 0.2% produced a greater inhibition than honey. The data on MIC suggest that the most susceptible micro-organism to honey is S. albus and the order of susceptibility is S. albus > S. aureus = P. aeruginosa >Klebsiella=Proteus > E. coli (Table 2). Candida albican was not susceptible to honey at all concentrations tested.

The pure honey was found to contain alkaloids, anthraquinone glycosides, cardiac glycosides, flavonoids, saponins, tannins and reducing compounds.

Table 1: Antibacterial activities of different concentrations of honey compared with ciprofloxacin and tetracycline

Organisms	Inhibition zone diameters (mm, mean values, N=3)
	Honey 100 %	Honey 50 %	Tetracycline  2.5%	Ciprofloxacin 0.2%
Staphylococcus albus	14.3	12.0	0	27.0
Staphylococcus aureus	11.0	0	10.0	30.0
Klebsiella species	12.3	8.0	6.0	29.7
Escherichia coli	13.3	0	16.0	27.5
Streptococcus faecalis	0	0	0	30.0
Pseudomonas aeruginosa	8.0	7.0	8.0	37.0
Proteus mirabilis	12.0	0	0	37.0

Table 2: Antibacterial activity of honey using the broth dilution method

Organisms	MIC (mg/ml)	MBC (mg/ml)
Staphylococcus albus	8.0	16.0
Staphylococcus aureus	32.0	64.0
Klebsiella species	128.0	128.0
Escherichia coli	128.0	256.0
Streptococcus faecalis	Undetermined	Undetermined
Pseudomonas aeruginosa	64.0	64.0
Proteus mirabilis	128.0	128.0

MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration

Discussion

Honey has been used to treat infections in a wide range of wound types, including leg ulcers, boils, pilonidal sinuses, and infected wounds from lower limb surgery.¹⁷

The present study showed that 100% honey inhibited growth of all the employed bacteria, both gram negative and gram positive in a dose- dependent manner, except with S. faecalis where there was no inhibition at all.  While ciprofloxacin (0.2%) was found to be more potent and more efficacious than honey, tetracycline at the concentration of 2.5% employed in this study was less potent except with E. coli where it was superior to honey. (Table 1). Furthermore, S. albus was the most susceptible to honey as indicated by the clear zone of inhibition obtained at 100% (v/v) as well as the MIC value.   This result compares well with the antibacterial action of manuka honey except that the latter had a greater potency.¹⁸

The following mechanisms have been suggested to explain the antimicrobial actions of honey;

1. Presence of an ‘inhibine’, factor in honey, which is hydrogen peroxide. ^{13, 19} Hydrogen peroxide is a well-known antimicrobial agent and its harmful effects when added in isolation is not noticeable with honey since the latter sequesters and inactivates the free iron which catalyses formation of oxygen free radicals produced by hydrogen peroxide. Its antioxidant components help to mop up free radicals. ⁹
2. Osmotic property: Honey being a super- saturated sugar exerts an osmotic pressure which makes little or no water available for the micro-organisms to survive. ²⁰
3. Stimulation of lymphocytic and phagocytic activity. ^{21, 22} Recent studies showed that the proliferation of peripheral blood B-lymphocytes and T-lymphocytes in cell culture is stimulated by honey a concentration as low as 0.1% and phagocytes are also activated by honey at such low concentrations. Furthermore, honey stimulates monocytes in cell culture to release cytokines, tumour necrosis factor (TNF) – alpha, interleukines (IL) -1 and (IL) –6, which stimulate the immune response to infection. In addition, the glucose content of honey and the acidic pH (typically between 3 and 4) may assist in the bacterial destroying action of macrophages.
4. Non- peroxide component: Among these are complex phenols and organic acids often referred to as flavonoids. ²³

While it could be said from the fore going that honey, when used in vivo might produce a greater effect than the in-vitro study, the antimicrobial profile might compare favourably with the present observation. However, the study has clearly demonstrated that honey might not adequately proffer a total solution to the current problems facing bacterial chemotherapy. Users therefore need to be enlightened that honey, being a natural product with very few side effects may not necessarily be superior to conventional therapies. The latter should be employed where necessary without skepticism.

Acknowledgement

The authors wish to appreciate the technical assistance of Mrs. O.O Ajala.

References

1. Gunther RT. The Greek herbal of dioscorides. Hafner, New York, 1968
2. Jeddar A, Kharsary A, Ramsaroop UG, Bhamjee A, Haffejee IE, Moosa A,  The antibacterial action of honey. An in-vitro study. S Afr Med J 1985;67: 257-258
3. Haffejee IE, Moosa A. Honey in the treatment of infantile gastro-enteritis. Br Med J 1985; 290: 1866-1867
4. Harris S. Honey for the treatment of superficial wounds: a case report and review. Primary Intention  1994; 2: 18-23
5. Vardi  A, Barzilay Z, Linder N, Cohen HA, Paret G, Barzilai A. Local application of honey for treatment of neonatal post-operative wound infection. Acta Paediatr 1998; 87: 429-432
6. Karayil S, DeshpandeSD, Koppikar GV. Effect of honey on multi-drug resistant organisms and its synergistic action with three common antibiotics. J Postgrad Med 1998; 44: 93-96
7. Helbling A, Peter C, Berchtold E, Bogdanor S, Muller U. Allergy to honey: relation to pollen and honey bee allergy. Allergy 1992;47: 41-49
8. Subrahmanyam M. Early tangential excision and skin grafting of moderate burns is superior to honey dressing: a prospective randomized trial. Burns 1999; 25: 729-731
9. Molan PC. Honey as a topical antibacterial agent for treatment of infected wounds. 11^th Conference of the European Wound Management Association, Dublin, 1991; 70
10. White JA. Composition of honey. In: Crane E (ed). Honey review. Heinemann, London,  1975;.157-206
11. Hunt TK. Recent advances in wound healing. Ann Surg 1970; 2: 1-10
12. White JW, Subers MH, Schepartz AI. The identification of inhibine. Am Bee J 1962;102: 430-431
13. White JW, Subers MH, Schepartz AI. The identification of inhibine. The antibacterial factor in honey as hydrogen peroxidase and its origin in a honey glucose oxygen system. Biochem Biophys Acta 1963;73: 57-70
14. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing a standardized single disk method. Am J Clin Pathol 1966; 45: 493-496
15. Barry AL, Thornsberry C. Susceptibility tests: diffusion test procedures. In: Lennette EA, Balows A, Hausller WJ, Shadomy HJ (eds). Manual of clinical microbiology. American Society for Microbiology, Washington, DC, 1985; 978-987
16. Farnsworth N. Biological and phytochemical screening of plants. J Pharm Sci 1966; 55: 225-276
17. Betts JA, Molan PC. A pilot trial of honey as a wound dressing has shown the importance of the way honey is applied to wounds. 11^th  Conference of the European Wound Management Association, Dublin, 2001
18. Cooper RA, Molan PC. The use of honey as an antiseptic in managing pseudomonas infection. J Wound Care 1999; 8: 161-164
19. Postmes T, Van den Bogaard AE, Hazen M. Honey for wounds, ulcers and skin graft preservation. Lancet 1993; 341: 756-757
20. Molan PC. The antibacterial activity of honey: 1. the nature of the antibacterial activity. Bee World 1992; 73: 5-28
21. Abuharfeil N, Al-Oran R, Abo-Shehada M. The effect of bee honey on the proliferative activity of human B- and T- lymphocytes and the activity of phagocytes. Food Agric Immunol 1999; 11: 169-77
22. Tonks A, Cooper RA, Price AJ, Molan PC, Jones KP. Stimulation of TNF-alpha release in monocytes by honey. Cytokine 2001; 14: 240-242
23. Molan PC, Russel KM. Non-peroxide antibacterial activity in some New Zealand honeys. J Apic Res 1988; 27: 62-67

Copyright 2006 - Annals of African Medicine