Antimicrobial Activities And Physico-chemical Analyses Of Honeys

1.5.6.4. As Immune Inducer

In vivo antibacterial activity of honey resulted in the induction of increased lymphocyte and phagocytic activity. Recent studies showed that the proliferation of peripheral blood B-lymphocytes and T-lymphocytes in cell culture is stimulated by honey at concentrations as low as 0.1% and phagocytes are activated by honey at concentrations as low as 0.1% (Halawani and Shohayeb, 2011). Honey at a concentration of 1% also stimulates monocytes in cell culture to release cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6, which activate the immune response to infection (Alvarez-Suarez et al., 2010; Tonks et  al., 2001; Tonks  et al., 2003).

It has been reported that Manuka honey increased IL- 1?, IL-6, and TNF-? production from Mono Mac6 cells or human monocytes,  and the  active component  was 5.8 kDa, which increased production of these cytokines via TLR4. In addition, it was reported that oral intake of  honey augmented  antibody  productions in  primary and secondary immune responses against thymus-dependent and thymus-independent antigens (Alvarez-Suarez et al., 2010).

1.5.6.5. As Anti-diabetic Agent

In the past, people with diabetes were advised to avoid “simple sugars” including honey. It was thought that consuming simple  sugars would cause a  sharp  and rapid elevation in blood glucose levels and an overwhelming  insulin  demand. Some even speculated that eating simple sugars could cause diabetes, a notion that has not been supported by scientific research (Al-Waili and Haq, 2004).

In fact, research has shown that some complex carbohydrates raise blood glucose levels more significantly than certain simple sugars. Both honey and sucrose have been shown to produce a lower glucose response  than starchy foods  such as  white bread. Moreover, it has been shown that the total amount of carbohydrate consumed is probably more important than the type of carbohydrate when it comes to blood sugar levels. Thus, experts agree  that  diabetics may include moderate amounts  of “simple sugars” in a balanced diet. Honey compared with dextrose caused a significantly lower rise in plasma glucose levels in diabetic subjects. It also caused reduction of blood lipids, homocysteine levels and CRP (C reactive protein) levels in normal and hyperlipidemic subjects (Tonks et al., 2007).

1.5.6.6. Antimutagenic and Antitumor Activity

Mutagenic substances act directly or indirectly by promoting mutations of the genetic structure. During the roasting and frying of food heterocyclic amines are  formed, e.g. Trp-p-1 (3- Amino-1,4-dimethyl-5H-pyridol [4,3-b] indole). The antimutagenic activity of honeys from seven different floral sources (acacia,  buckwheat, fireweed, soybean, tupelo and Christmas berry) against Trp-p-1 was tested by the Ames assay and compared to a sugar analogue as well as to individually tested simple sugars (Wang et al., 2002). All honeys exhibited a significant inhibition of Trp-p-1 mutagenicity. Glucose and fructose were found to have  a  similar antimutagenic activity as honey. Nigerose, another sugar, present in honey has an immunoprotective activity (Murosaki et al., 2002). The antimetastatic effect of honey and its possible mode of anti-tumor action was studied by the application of honey in spontaneous mammary carcinoma in methylcholanthrene-induced fibrosarcoma  of  CBA mice and in anaplastic colon adenocarcinoma of Y59 rats (Orsolic and Basic, 2004). A statistically significant anti-metastatic effect was achieved  by  oral  application of honey (Orsolic et al., 2003). These findings indicate  that  honey  activates the immune system and  honey ingestion  may be advantageous with  respect to cancer and metastasis prevention.

In addition, it is postulated that honey given orally before tumour cell inoculation may have a decreased effect on tumour spreading. In another study of the same group the effect of honey on tumour  growth,  metastasising  activity  and induction of apoptosis and necrosis in murine tumour models (mammary and colon carcinoma) was investigated. A pronounced antimetastatic effect was observed when honey was applied before tumour-cell inoculation (per oral 2 g kg-1 for mice or 1 g  kg-1 for rats, once a day for 10 consecutive days) (Swellam et al., 2003).

In another study the anti-tumour effect of honey against bladder cancer was examined in vitro and in vivo in mice. According to these results honey is an effective agent for inhibiting the growth of different bladder cancer cell lines (T24, RT4, 253J  and MBT-2) in vitro. It is also effective when administered intralesionally or orally in the MBT-2 bladder cancer implantation mice models (Swellam et al., 2003).

1.5.6.7. As Treatment for Arthritis

Apparently also boron stimulates in a positive way, hormonal factors for both men and women, resulting in healthy bones. If this hormonal balance is disturbed, it

will lead to osteoarthritis and as honey contains boron, it’s routinely consumption can avoid such problems (Bingham et al., 2010).

1.5.6.8. As  Skin Disinfectant

Typical honeys are about eight times more potent against coagulase-negative staphylococci than if bacterial inhibition were due to  their  osmolarity  alone. Therefore, honey applied to skin at the insertion points of medical devices may have a role in the treatment or prevention of infections by coagulase-negative staphylococci (French et al., 2005).

1.5.6.9. The Action of Honey in Wound Healing

Honey is an effective treatment of wounds because it is non-irritating, non- toxic, self-sterile, bactericidal, nutritive, easily applied and more  comfortable  than other dressings (Singh et al., 2012). The treatment of wounds with honey has rendered them bacteriologically sterile within 7-10 days of the start of the treatment and  promoted healthy granulation of tissue according to Tan et al.(2009)

Anti-fungal activity of honey has been also tested on Candida albicans, C. pseudotropicalis, C. stellatoidea and C. tropicalis and all were found to be susceptible (Akujobi and Njoku, 2010; Anyanwu, 2012). Honey was also found to be more  effective as an antibacterial agent against several Pseudomonas and Staphylococcus strains than the antibiotic, gentamicin (Tan et al., 2009).

In a clinical study involving 59 patients with wounds and  ulcers,  most  of which had failed to respond to conventional treatments, 15-30 ml fresh honey was applied daliy. The bacteria isolated from 58 of these wounds (E. coli, S. aureus, P. mirabilis, mixed coliforms, Klebsiella species, and S. faecalis) were all susceptible to honey in vitro according to Tan et al. (2009). One other  bacteria, Ps.  pyocyanea did  not undergo complete lysis in vitro tests but it was completely sterilised in vivo. In     one case in which the patient had a buruli ulcer infected  with  Mycobacterium  ulcerans, honey treatment was ineffective an in vitro tests showed the mycobacteria to be resistant to honey (Subrahmanyam, 2007).

Honey has cleansing action of wounds, stimulates tissue regeneration, reduces inflammation and honey impregnated pads act as non-adhesive tissue  dressing (Singh  et al., 2012).

1.5.7. Factors that Affect Antimicrobial Activity of Honey

The instability of honey inhibine was first recognized by Dold, who found that  it was destroyed by heating and by exposure to light. These observations have since

been confirmed by numerous other researchers, but there have been differences in the degree of instability reported (Moussa et al., 2012; Chen et al., 2012).

a. Sensitivity to heat

According to Chen et al. (2012) and Hassan (2013), the reporton the loss of  antibacterial activity on exposure of honey to heat was of complete loss of  inhibition  by 17% honey after exposure of 50% honey to 100°C for 5 min, 80°C for 10 min, or 56°C for 30 min. However, this did not mean that antibacterial activity was lost completely: if the unheated honey had been of just high enough activity to inhibit growth when at 17%, not much activity would have to be lost on heating for inhibition no longer to be seen. This also applies to the similar finding of  Pothmane  that  exposure of honey to 100°C for 5 min or 56°C for 1 h caused complete loss  of inhibition by 17% honey. In later reports the researchers used a dilution series for the assay of activity. Although complete loss of inhibition in their studies still did  not  mean that antibacterial activity was lost completely, its reduction to a level below detestability would generally represent a loss of  80% or more, if not a complete loss.   In these reports 'complete loss' was found to result from exposure of honey to: 100°C  for 30 minutes; 100°C for 15 min (Schade et al.,1958; Hassan, 2013); 90°C for 8 minutes ( Gryuner and Arinkia, 1970); 100°C for 5 min, 90°C for 15 min, 70-80°C for 20-30 min, and 56°C for 60 min (Franco and Sartori, 1940); 80°C for 15 min (Stomso-Stitz and Kominos, 1960); 80°C for 30 min (Ialomitzeanu and Daghie 1973); 60°C for 15 min and from use of 'heated honey' (Dustmann, 1987). An  almost  complete loss was found on heating honey for 100°C for 10  min  (Chambonnaud, 1966).  In another report the activity was not lost completely after exposure  of honey   to 100°C for 15 min, but was reduced to the same level as that of artificial honey, indicating that all activity other than that due to osmolarity had been destroyed  (Christov and Mladenov, 1961). A similar finding was made with honey boiled for 10 min (Plachy, 1944). Others also have found that only part of the antibacterial activity    is destroyed by heating honey. Exposure of honey to 100°C for 10 min  caused  complete loss of activity against seven species of bacteria, but only partial loss of activity against Bacillus pumilus and a strain f Streptomyces, and no loss of activity against Bacillus subtilis and Sarcina lutee. Another report about half of the activity against B. subtilis was found to be heat-stable (Gonnet and Lavie,  1960).  Heating honey at 56°C for 30 min caused a loss of activity that was  greater against some  species than against others'. The presence of both heat-stable and heat-sensitive factors has been reported by others also (Chambonnaud, 1968; Gryuner and Arinkia, 1970; Daghie et al., 1971; Bogdanov, 1983). The retention of part of the activity reported in instances where honey has been subjected to lesser degrees of heating probably results from there being only partial destruction of the heat-sensitive factor, rather than a heat-stable factor being responsible (Moussa et al., 2012).

According to Chen et al. (2012), the minimum inhibitory concentration of honey was found to increase from 4% to 8% after exposure of honey to 46°C for 8 h, to 12%     after exposure to 52°C for 8 h, and to 16% after exposure of honey to 55°C for 8 h.  Also reported was complete loss of activity after exposure to more than 65°C for less than 4 h, a heavy but not complete loss after exposure to 56°C for 24 h, but no loss   after exposure to 40°C for 96 h (Moussa et al., 2012).

The stability of the antibacterial activity in heated honey has been found to depend on the pH, activity being more rapidly lost at low pH according to Moussa  et  al. (2012). According to Singh et al. (2012), there are some large differences in the findings on the stability of the antibacterial activity of honey at lower temperatures,    but generally the conclusion has been that it is stable below 40°C. No decrease in antibacterial activity was seen in honeys held at 40°C for 96 h, as in the  case  mentioned above, nor in honey held at 37°C for 24 h. This is to be expected when it is borne in mind that the temperature in the beehive where honey can spend quite a long time is around 34°C. It  may not be as stable at this temperature when diluted: the rate  of production of hydrogen peroxide drops off with time, and the amount of hydrogen peroxide present after 16 h was found to be much lower than that present after the first hour (Moussa et al., 2012). Others have also reported that honey is less stable when diluted. This could be a consequence of the build-up of gluconic acid, or of damage to the glucose oxidase from free radicals generated from hydrogen peroxide as discussed earlier (Hasan, 2013). The latter suggestion is supported by the finding with  the  isolated enzyme that addition of a high level of hydrogen peroxide inactivated it after about 30 min. However, it has been reported that 50% honey  held  at  room  temperature for 100 h does not lose its antibacterial activity (Molen, 2007). There are several indications of the antibacterial activity of honey being very stable at room temperature.

b. Sensitivity to light

It has been known since some of the earliest work on  the  antibacterial properties of honey that the activity is unstable in light. Chen et al. (2012) reported

that honey lost its ability to inhibit bacterial growth (tested in a 17% solution) after exposing a thin film of it to sunlight. Others have since confirmed this observation according to Chen et al. (2012). Exposure of honey in a layer 1-2  mm  thick  to  sunlight for 15 min was found to result in complete  loss  of  non-osmotic  activity. When not spread out in a thin layer it has not been found to be so sensitive: almost complete loss of activity after 18 days in direct sunlight, gradual disappearance of activity when exposed to direct sunlight but not with diffuse  daylight,  and  a  significant reduction in activity in honey samples stored for 3-6 months on  open  shelves (more than twice that lost in the same samples stored in a dark cupboard) have been reported (Tan et al., 2009). No loss of activity was found, however, when a thin film of honey was exposed for 1 h to an ultraviolet (UV) lamp (254  nm) (Molen,  2002).

A large loss of activity was found in honey left for 8 months on a window-sill  on the sunny side of the building if stored in 1 or 2.5 litre jars made from clear polystyrene, but not if stored in jars made of white or ivory polyethylene with low transmission of light of wavelength below 400 nm. Glass jars coated with a film to absorb UV light were only partially successful in this study in preventing the loss of activity, indicating the necessity to protect from light of wavelengths up to 400 nm (Mandel and Mandel, 2011).  This protection by absorption of light can occur within  the honey itself, as is seen with the greater stability of bulk quantities compared with thin films. Dark-coloured honey was found to be more light-stable than light-coloured honey,  presumably because it is less light into the bulk of the  honey (Alvarez-Suarez  et al., 2013). However, the sensitivity to light has been observed to depend on  the  floral source of the honey: in a 500 g jar kept in sunlight, some floral types of honey were found to lose their activity completely in only 48 h, and a reduction of up to 67% in the production of hydrogen peroxide (Hasan, 2013)