The Honey, As the First Sweetening Agent Known to Man, Has a Long and Interesting History.1In

INTRODUCTION

The Honey, as the first sweetening agent known to man, has a long and interesting history.1In addition to use in food, its origin, availability and properties are responsible for its wide variety of services to ancient and medieval man.2 The history of the use of Honey is parallel to the history of man. Honey is the natural sweet substance produced by honeybees from the nectar of blossoms which honeybees collect, transform and combine with specific substances of their own, store & leave it in the Honeycomb to ripen & mature.3Honey is prepared by bees from plant nectars, from plant secretions and from excretions of plant sucking insects. The Food Standards Code defines it as “the nectar and saccharine exudations of plants gathered, modified and stored by the Honeybee”.4

Honey has been used as a medicine since ancient times and is still used in folk medicine.2 Ayurveda, the traditional Indian system of medicine, describes Honey as the nectar of life.5 It employs Honey predominantly as a vehicle for faster absorption of various drugs such as herbal extracts.6 Greek and Roman medicine employed Honey for a multitude of purposes.1

Thirty percent of the 966 prescriptions in the ancient Egyptian Papyrus Ebners (1550 BC) used Honey.1 The ancient physicians were aware of the differences in the therapeutic value of the Honeys available to them. Aristotle (384-322 BC) discussing differences in Honey, referred to pale Honey being good as a salve for sore eyes and wounds7and Dioscorides (C.50 AD) stated that a pale yellow Honey from Attica was the best ,being good for all rotten and hollow ulcers.8Honey has been a valued part of wound treatment for many centuries. It was first documented as a wound treatment by the Egyptians in 2000 BC.9Russian soldiers in World War 1 used Honey, apparently successfully, for wound healing purpose.1

Beck & Smedley summarised the traditional uses of Honey as follows: “… its main employment was as a helpful remedy for gastric and intestinal disorders, especially as a pleasant laxative. Respiratory troubles were next in order. The sedative and soporific power of Honey is often emphasized. The diuretic effect of Honey was well known and it was a favored remedy for all kinds of inflammation of the kidneys, for gravel and for stones. The antiseptic property of Honey made it a desirable gargle, expectorant and a valuable adjunct in mouth hygiene. In inflammation of the eyes and eyelids Honey was extensively used. In surgical dressings and skin diseases it was a remedy of first choice. The smallpox patients were anointed with Honey. It was also employed as a vehicle for nauseous or bitter medicines”.10

Honey is also known to improve food assimilation and to be useful for chronic and infective intestinal disorders such as constipation, duodenal ulcers and liver disturbances. Honey is well known remedy for colds and mouth, throat or bronchial irritations and infections. It reduces and cures eye cataracts, cures conjunctivitis and various afflictions of the cornea.3 It has been used in a wide range of conditions including skin, eye, respiratory and gastro-intestinal illnesses. Honey also has therapeutic features that indicate it has potential for the treatment of periodontal disease as well as for cleaning infection in mouth ulcers and wounds from oral surgery.11Honey has the potential to use in the treatment of skin infections, gastrointestinal conditions and eye infections.3Honey may be used alone or in combination with other substances, and has been administered both orally and topically.

Although it has been used as a medicine since ancient time in many cultures, there was no recognition of its antibacterial properties, it was just known to be an effective remedy.12In 1989, an editorial in the Journal of the Royal Society of Medicine expressed the opinion: "The therapeutic potential of uncontaminated, pure Honey is grossly underutilized. It is widely available in most communities and although the mechanism of action of several of its properties remains obscure and needs further investigation, the time has now come for conventional medicine to lift the blinds off this 'traditional remedy' and give it its due recognition".13

Now it can be seen that the effectiveness of Honey in many of its medical uses is probably due to its antibacterial activity. Honey has been demonstrated in many studies to have antibacterial effects, attributed to its high osmolarity, low pH, hydrogen peroxide content and other uncharacterized compounds.It is well established that Honey inhibits a broad spectrum of bacterial species. .14

The medicinal use of Honey in wound treatment is derived from diverse ancient civilizations.15In recent times, the use of Honey as a wound dressing material, an ancient remedy, has been rediscovered.13Numerous reports of animal models, clinical studies, case reports and randomized controlled trails have shown that it rates favorably alongside modern dressing materials in its effectiveness in managing wounds.16,17 Honey has a potent antibacterial activity and is very effective in clearing infection in wounds18 and protecting wounds from being infected.17 Honey also provides rapid autolytic debridement and deodorizes wounds.19It has been observed that Honey reduces inflammation and pain, causes oedema and exudation to subside and increases the rate of healing by stimulation of angiogenesis, granulation and epithelisation.20,21The rapid clearance of infection is the most notable feature of Honey as a wound dressing, in many cases when no conventional therapy had worked.18,22 Unlike other antiseptics, Honey is not cytotoxic so it does not slow healing, nor does it have any adverse side effects like antibiotics do.22,23

Honey has been used as an excellent adjuvant for accelerating wound healing and has received wide acceptance.17,18,20 Topically applied Honey has been tried in the treatment of burn wounds in some recent clinical trails and the results are promising.5,21,22,24 Honey’s anti-inflammatory action is thought to reduce the damage caused by the free radicals arising from the inflammation, thus preventing further necrosis.5 It has been observed that Honey prevents partial thickness burns from converting to full thickness burns that require skin grafts.25

Honey is a product with minimal types and levels of microbes; this is attributable to the natural properties of Honey and control measures in the Honey industry.4,26 However, Honey can carry yeasts and spore forming bacteria and hence, Honey and the product in which Honey is used, can be inadvertently inoculated with undesirable microbes.27,28 Bacterial spores, particularly those in Bacillus genus are commonly found in Honey and Clostridial spores are also sometimes found.29,30,31,32Clostridium botulinum is known to cause botulism and infant botulism.33

As Honey can sometimes contain spores of Clostridium botulinum and other microorganisms, there is a definite risk of introducing the microorganisms into the wounds, if Honey is used as a dressing. Therefore, its use as a wound dressing has been argued, however on the ground that the risk of it possibly causing wound botulism is unacceptable.34

It is worth considering whether Honey can be sterilized for use as a dressing as it could be considered negligent to dress a wound with Honey that has not been subjected to a quality assurance procedure or effective sterilization procedure to guarantee the absence of pathogens. If Honey could be sterilized for use as a wound dressing this would remove the risk. Hence the sterility of Honey is important.

Continued use of some systemic and topical antimicrobial agents has imparted the selective pressure and led to the emergence of antibiotic resistant strains in a wide variety of nosocomial infections worldwide. The evolution of selective pressures in hospitals andcommunities alsofacilitate the development andspread of multiresistant organisms.35Thus MRSA, ESBLs and resistance of other organisms to antimicrobial agents is an increasing problem in many areas of the world especially in developing countries. This in turn has driven the continued search for new agents for the treatment of infections. Unfortunately, the increased cost of searching for such new agents and the decreasing rate of their discovery36has made the situation increasingly urgent to find alternative options of treatment.

The prevalence of multidrug resistant organisms now justifies the interest in re-evaluation of traditional remedies37 and the general ‘back to nature’ trend in solving these problems with indigenous knowledge. The use of traditional medicine has been practiced since the origin of mankind, and in the past it was the only method available. The integration of traditional and modern medicine is gaining increased recognition globally. Any possible remedy that is cheap, non toxic and unlikely to select for further antibiotic resistant strains merit investigation and hence Honey can be studied for its use as an alternative therapy.

Some authors have worked on the antibacterial activity of Indian honeys but alittle account was taken of the variety of different honey samples available in India. There is a vast variety of honey samples available according to its floral source. As mentioned earlier honey is also used in India for a multitude of purposes but importance is not given to the type of honey used. It would be of great help if the honey possessing highest antibacterial activity were used for any medicinal purpose. Hence, there is a need for exploring different Indian honey samples available for their level of antibacterial activity.

Several brands of honey with standardized levels of antibacterial activity are commercially available in Australia and New Zealand. P. C. Molan, Honey Research Unit, University of Waikato, Hamilton, New Zealand has done extensive study on Manuka honey. Bees gather manuka honey from the nectar of the flowers of the manuka bush (Leptospermum scoparium), a native plant that grows in the remote unspoilt areas of New Zealand. Dr Peter Molan and his research team at the Honey Research Unit found in some specific strains of manuka honey a second natural more powerful and more stable antibacterial property called UMF (Unique Manuka Factor). UMF is additional to the hydrogen peroxide activity and gives the honey a wider range of uses and effectiveness.38

In laboratory tests the UMF property has been found to be effective against a wide range of bacteria including Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes. Manuka honey has been useful in the treatment of skin ulcers from menginoccal septicaemia, infected surgical wounds, and abscess.39 Manuka honey is well known for its medicinal properties, as it is a commercially available standardized honey. Therefore, considering Manuka honey as a gold standard honey, Indian honeys can be explored for their antibacterial activity.

AIMS & OBJECTIVES

Hence, the present study was conducted to study the antibacterial activity of various Indian Honey samples and to evaluate an effective method to sterilize honey.

The present study has been planned with followingAims and Objectives.

Phase I

To isolate & identify following organisms from different samples such as wound, pus, sputum etc.
E. coli
Klebsiella sp.
Pseudomonas sp.
Staphylococcus aureus

To study Antibiotic sensitivity pattern of these isolates to a standard set of antibiotics by Kirbey-Bauer disc diffusion method.

To categorize the organisms as sensitive, MDR & partially sensitive (Non-MDR).

Phase II

Collection of various Indian Honeys from various sources.

To procure Manuka Honey (UMF 16 +)from New Zealand to compare with Indian Honey samples by all aspects. (Manuka honey is considered as Gold Standard as it has been proven for it’s antibacterial activity by Dr. Molan from New Zealand)

To perform sterility testing of collected Indian Honeys along with Manuka honey.

Physicochemical & biochemical analysis of different types of Indian Honeys & Gold Standard Manuka honey from New Zealand & compare.

To determine the enzyme levels (glucose oxidase, catalase, diastase) from various Indian Honeys & Manuka honey from New Zealand & compare.

Phase III

To determine the in vitro antimicrobial effect of various Honey samples by agar well diffusion method.& compare with Gold standard Manuka honey from New Zealand.

To determine Minimum Inhibitory Concentration of Honey for isolated organisms by Agar dilution method.& compare with Gold standard Manuka honey from New Zealand

To compare the antimicrobial effect of Honey with that of antibiotics on isolated organisms.

To determine the synergistic action of honey & antibiotics.
To check the effect of 6 months storage of Honey samples at room temperature for its physicochemical parameters, enzyme levels& antimicrobial activity.

METHODOLOGY & OBSERVATIONS

The present study was carried out during the period February 2005 to February 2006, in the Department of Microbiology, at T.N.MedicalCollege, B.Y.L.Nairch.Hospital, Mumbai Central, Mumbai 400008.

The study was planned in III phases. All results are statistically evaluated.

Phase I:Organisms -

Total 1520 clinical samples (wound swab, pus swab, pus & sputum) were processed for bacterial study. Bacterial growth was observed in 607 clinical samples.

Staphylococcus aureus, Escherichia coli, Klebsiella sppPseudomonas aeruginosa were selected for present study. Each isolate was identified & speciated by microscopy, culture & biochemical tests as shown in Photograph 1, 2& 3.

Total 400 isolates were obtained. Each organisms was further categorized as MDR, Non-MDR, &Sensitive, according to their antibiotic sensitivity pattern40,

as shown in Photograph 4

MDR strains were resistant to all 6 antibiotics.

Non-MDR(NMDR) strains were sensitive to at least 2 antibiotics.

Sensitive strains were sensitive to all 6 antibiotics.

All 400 isolates were checked for 6 antibiotics along with ATCC strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and

Pseudomonas aeruginosa ATCC 27853)

Table 1 & Graph 1 shows distribution of organisms. Maximum MDR strains were observed in Pseudomonas aeruginosa.

Table 1: Distribution of organisms

E. coli

Klebsiella spps

S. aureus

P. aeruginosa

Total

MDR

(26.32) /

(25.88) /

(21.92) /

(34.38) /

112

(28.00)

NMDR

(65.79) /

(64.71) /

(65.75) /

(57.03) /

251

(62.75)

Sensitive

(7.89) /

(9.41) /

(12.33) /

(8.59) /

(9.25)

Total

114

(100.00) /

128

(100.00) /

400

(100.00)
Chi-square Test
applied /  2 value / Df / P-value / Association is-
5.722 / 8 / 0.6783 / Not significant

As apparent in the above table, Ps. aeruginosa showed highest MDR (34.38%),

E.coli had maximum NMDR (65.79%) and S. aureushad maximum sensitive

strains (12.33%).

However, comparison of MDR, NMDR and sensitive showed no statistical significant association with type of Organism (P-value=0.6783).

Graph no. 1

Table 2 & Graph 2 explains MDR organisms isolated from various samples.

Table No. 2: MDR organisms isolated from various clinical samples.

Sr.
No. / Organism / Clinical samples / Total
Wound swab / Pus swab / Pus / Sputum
1. /

Escherichia coli

/ 11
(36.60) / 5
(16.70) / 6
(20.00) / 8
(26.70) / 30
(100.00)
2. / Klebsiella species / 9
(40.90) / 5
(22.72) / 4
(18.19) / 4
(18.19) / 22
(100.00)
3. / Staphylococcus aureus / 5
(31.25) / 3
(18.75) / 5
(31.25) / 3
(18.75) / 16
(100.00)
4. / Pseudomonas aeruginosa / 21
(47.73) / 5
(11.36) / 8
(18.18) / 10
(22.73) / 44
(100.00)
Total / 46
(41.07) / 18
(16.08) / 23
(20.53) / 25
(22.32) / 112
(100.00)

Key: ( ) = %

Chi-square Test
Applied /  2 value / Df / P-value / Association is-
3.877 / 9 / 0.9193 / Not significant

The apparent difference in occurrence of different MDR organisms in various clinical samples failed to show statistically significant association (P-value=0.9193), indicating that the occurrence of various MDR organisms does not differ significantly between various clinical samples.

Table 3 & Graph 3 gives sensitive organisms isolated from various clinical samples.

Table No. 3: Sensitive organisms isolated from various clinical samples.

Sr.
No. / Organism / Clinical samples / Total
Wound swab / Pus swab / Pus / Sputum
1. /

Escherichia coli

/ 4
(44.40) / 0
(0.0) / 2
(22.20) / 3
(33.4) / 9
(100.00)
2. / Klebsiella species / 3
(37.50) / 2
(25.00) / 1
(12.5) / 2
(25.00) / 8
(100.00)
3. / Staphylococcus aureus / 3
(33.33) / 1
(11.12) / 3
(33.33) / 2
(22.22) / 9
(100.00)
4. / Pseudomonas aeruginosa / 6
(54.54) / 0
(0.0) / 1
(9.10) / 4
(36.36) / 11
(100.00)
Total / 16
(43.24) / 3
(8.10) / 7
(18.91) / 11
(29.72) / 37
(100.00)

Key: ( ) = %

Chi-square Test
Applied /  2 value / Df / P-value / Association is-
7.340 / 9 / 0.6018 / Not significant

As with the MDR organisms, the occurrence of different sensitive organisms in various clinical samples did not show statistically significant association

(P-value=0.6018) with the different clinical samples, indicating that the occurrence of various sensitive organisms does not differ significantly between various clinical samples.

Table 4 & Graph 4 gives an account of Non MDR strains isolated from various clinical samples.

Table No. 4: Non-MDR organisms isolated from various clinical samples.

Sr.
No. / Organism / Clinical samples / Total
Wound swab / Pus swab / Pus / Sputum
1. /

Escherichia coli

/ 21
(28.00) / 8
(10.60) / 28
(37.40) / 18
(24.00) / 75
(100.00)
2. / Klebsiella species / 23
(41.81) / 8
(14.55) / 14
(25.46) / 10
(18.18) / 55
(100.00)
3. / Staphylococcus aureus / 11
(22.92) / 7
(14.58) / 19
(39.58) / 11
(22.92) / 48
(100.00)
4. / Pseudomonas aeruginosa / 39
(53.43) / 5
(6.85) / 12
(16.43) / 17
(23.29) / 73
(100.00)
Total / 94
(37.45) / 28
(11.15) / 73
(29.08) / 56
(22.31) / 251
(100.00)

Key: ( ) = %

Chi-square Test
Applied /  2 value / Df / P-value / Association is-
20.444 / 9 / 0.0154 / Significant

In contrast to occurrence of MDR organisms and Sensitive organisms in various clinical samples, the occurrence of different NMDR organisms in various clinical samples was found to have statistically significant association (P-value=0.0154) with the different clinical samples, indicating that the proportion of occurrence of various NMDR organisms did differ significantly between various clinical samples.

Phase II:Honey

Total nine Indian honey samples along with Manuka (UMF 16+) –New Zealand were studied for their different parameters.

Manuka honey (UMF 16+) is one of the New Zealand honey extensively studied by Dr. Molan for its antibacterial properties & medicinal uses. It was procured from New Zealand & compared in all aspects with all nine Indian honey samples.

I) The different types of Indian honey used for the study are as follows -

Type I – Unifloral honey

a)Jambhul honey (Phondaghat Pharmacy)

b)Eucalyptus honey (Phondaghat Pharmacy)

c)Mustard honey (CBRTI- Pune)

Botanical names

Jambhul – Syzygium cumini

Eucalyptus – Eucalyptus globules

Mustard – Brassica campestris

Type II – Multifloral honey

a)From Kashmir (CBRTI- Pune)

b)From Maharashtra (Phondaghat Pharmacy)

c)From Kothimbe (Vanvasi Ashram)

Type III – Branded honey marketed by

a)Dabur

b)Phondaghat Pharmacy

c)Khadi & Village Industries Commission

Type IV – Manuka Honey (UMF 16+) from New Zealand

Manuka honey was considered as Gold Standard & all parameters of nine Indian honey samples were compared with it.

All nine Indian honey samples & New Zealand Manuka honey were studied for their

Physicochemical properties
Enzyme levels
Sterility testing
Antibacterial activity by

a)Agar well diffusion method

b)Agar dilution method

Synergistic action of honey with respective antibiotics

All parameters were studied again after six months with all honey samples to check the effect of aging on honey. All honey samples were stored at room temperature in dark.

1. Physicochemical properties

The physicochemical analysis of honey samples was carried out at S. S. Laboratories, Mumbai using standard methods. The parameters analyzed were as follows:

Color, pH, specific gravity, ash, moisture, sucrose, total reducing sugars, glucose, Fructose/glucose ratio, acidity as formic acid, Fiehe’s test & Aniline chloride test.

Table no. 5: The physico-chemical properties of the honey samples

Honey / Color / pH / Specific gravity / Sucrose % / Ash % / Moisture % / Total reducing sugars % / Fructose/
Glucose ratio / Acidity Formic acid % / Fieches test / Aniline chloride test / Glucose %
Jambhul / Dark brown / 3.2 / 1.390 / 1.27 / 0.20 / 20.4 / 66.95 / 1.129 / 0.40 / + / - / 32.74
Eucalyptus / Brown / 3.9 / 1.389 / 0.88 / 0.21 / 20.0 / 72.45 / 1.0375 / 0.120 / + / - / 36.97
Mustard / Brown / 4.1 / 1.390 / 2.22 / 0.20 / 20.2 / 71.55 / 1.0120 / 0.119 / + / - / 36.60
Kashmir / Brown / 4.2 / 1.387 / 3.22 / 0.23 / 20.0 / 69.86 / 1.0525 / 0.122 / + / - / 35.40
Maharashtra / Dark brown / 3.5 / 1.390 / 1.52 / 0.20 / 20.3 / 71.35 / 0.95 / 0.21 / + / - / 38.05
Kothimbe / Brown / 4.2 / 1.370 / 1.4 / 0.21 / 20.4 / 70.02 / 0.98 / 0.13 / + / - / 36.7
Dabur / Brown / 4.5 / 1.387 / 1.05 / 0.23 / 20.2 / 72.93 / 0.9775 / 0.118 / + / - / 38.30
Phondaghat / Brown / 4.4 / 1.382 / 1.02 / 0.20 / 20.6 / 71.58 / 0.99 / 0.123 / + / - / 34.2
Khadi / Brown / 4.1 / 1.364 / 1.7 / 0.22 / 20.2 / 70.20 / 1.02 / 0.116 / + / - / 35.7
Manuka / Brown / 3.4 / 1.398 / 1.62 / 0.23 / 20.0 / 71.80 / 1.1853 / 0.122 / + / - / 34.25

No drastic change was observed in physicochemical properties of honey samples after 6 months of storage.