Antimicrobial Activities And Physico-chemical Analyses Of Honeys

1.5.4. Factors contributing to Antimicrobial Properties of Honey

Until 1963, it was thought that the antimicrobial properties of honey were mainly because of hydrogen peroxide, but further studies have indicated that other physical factors like acidity, osmolarity (Molan, 1992) and electrical conductivity, and chemical factors including volatile compounds (Yao et al., 2003),  antioxidant  (Gheldoff et al., 2002; Henriques et al., 2006), beeswax, propolis and pollen (Viuda- Martos et al., 2008) play a considerable role in antimicrobial activity.

1.5.4.1. Osmotic Effect

Honey is a super saturated solution of sugar (80%) and water (17%). The osmolarity of honey inhibits microbial growth because of the  strong interaction of  sugar molecules with water molecules thus; insufficient water molecules are available  to support microbial growth. This availability is known as water activity (aw). Water involved in many metabolic processes in many organisms. Depending on the permeability of cell membrane in each organism the water activity (aw) of many bacterial species vary between 0.94-0.99. The  water activity of  honey is  0.6 because  of high sugar molecules and low water thus many species cannot grow in that environment. Fungi can tolerate a lower aw than bacteria, so reports of antifungal activity with diluted honey reveal that there are more factors involved than only the sugar content of honey. Also, Staph aureus has a high tolerance of low aw (0.86) ie. can tolerate high NaCl level but not high sugar therefore, it is considered as one of the most susceptible species to the antibacterial activity of honey (Molan, 1992).

1.5.4.2. Acidity

Honey is quite acidic; normally, it has an average pH of 3.9 (with a typical  range of 3.2 to 4.5). It has been known that this acidity is a result of the conversion of glucose to gluconic acid with help of glucose oxidase enzyme (Molan, 2001). The optimum pH for growth of many bacterial species is 7.2 –  7.4.  However, the  lowest pH value for growth of some wound pathogens is 4.3 for E .coli and 4.4 for P. aeruginosa. The low pH of honey is therefore important to slow down or inhibit bacterial growth (Bogdanov,2009; Molan, 2000). Since 2001, the osmotic effect was thought to be the main factor for antimicrobial activity (Molan, 2001). However, in  2005 a study compared honey and sugar solution of same osmotic effect on coagulase negative staphylococci. The study confirmed that antimicrobial properties are not exclusively due to osmotic effect (French et al.,  2005).  It has been noted that  the pH  of honey also generates and maintains good environment for fibroblast activity (Lusby et al., 2002).

1.5.4.3. Hydrogen Peroxide Production

In 1919, Sackett reported that in diluted honey the antibacterial properties of honey were increased. This is because when honey is diluted, hydrogen peroxide is released with the help of an enzyme (glucose oxidase) that is found in honey (Molan, 1992). This enzyme is secreted by the hypopharyngeal gland of bees and added to  nectar during honey formation (Alvarez-Suarez et al., 2013).

Glucose + H2O + O2 glucose oxidase Gluconic acid + H2O2

Hydrogen peroxide (H2O2) is considered to be one of the main factors in antibacterial activity of honey. It is involved in cell multiplication in  different  cell types in the body as certain concentration of H2O2 can support epithelial cells and fibroblast growth to repair damage or injury (Burdon, 1995). It also promotes wound healing by regeneration of new capillaries (Tur et al., 1995). The enzyme (glucose oxidase) is inactive in full strength honey due to the low pH, so the diluting action of fluids produced by the wound is thought to activate glucose oxidase to produce hydrogen peroxide. In addition, it stays in the honey during storage without losing activity. Hydrogen peroxide was used for long time to disinfect wounds in hospitals. This chemical causes damage to the tissues and inflammation  due to  free radical that   is produced. The levels of H2O2 in honey are around 1000 times lower than those applied as antiseptic on wounds (Molan, 2001). As a result it does  not  inflame  a wound or damage the tissue (Bang et al., 2003). Weston (2000)  suggested  that  the level of H2O2 was related to floral source, and that it depended  on  the  balance  between the production and destruction rate of H2O2. Destruction of H2O2 is due to catalase which derives from  both the pollen and the  nectar of plants,  and the amount  of catalase in different sources is variable. In addition, Brudzynski (2006) studied the effect of H2O2 on the antibacterial activity of 42 honey samples from Canada. She  found that the antibacterial activity was correlated with production of H2O2 in honey.

1.5.4.4.Non-Peroxide Components

Several efforts were made to identify the non-peroxide  antibacterial  components present in the honey (Allen et al., 1991). Weston et al.,  (1999) separated the antibacterial phenolic fraction (APF) from the honey which consisted of benzonic acids, cinnamic acids and flavonoids. It was determined that APF plays a small role in manuka honey as non-peroxide antibacterial component, therefore, there are other factors which were need to be identified. Honey contains a variety of polyphenolic compounds that may be capable of chelating metal ions and decreasing oxidation (Gheldof et al., 2002). Two important classes of phenolic compounds are flavonoids  and phenolic acid which are known as  natural antioxidants (Molan,  1992; Pyrznska  and Biesaga, 2009; Yao et al., 2003). In a study performed by Wahdan (1998), two phenolic acids were extracted for the first time; these  were caffeic  acid and ferulic  acid. Flavonoids had shown a range of biochemical and pharmacological  actions,  which affect the inflammatory cells and the generation of inflammatory processes (Viuda-Martos et al.,  2008). The use of flavonoids in medicine is increasing due to  their ability to trap free radicals, to stimulate hormones and neurotransmitters, and to inhibit specific enzymes which produce superoxide anions (Pyrznska and Biesaga, 2009).

However, it has been identified that several organic components in the ether extract of honey possess antibacterial activity; these include 3,5-dimethoxy-4-hydroxy benzoic acid (syringic acid), and methyl 3,5-dimethoxy-4- hydroxy benzoate (methyl syringate) (Alvarez-Suarez et al., 2013). By using high performance liquid chromatography (HPLC), some other flavonoids and phenolic acids have also been identified in different honeys, for example, pinocembrin, pinobanksin and chrysin

(Bogdanove et al., 1989), gallic acid and abscisic acid (Yao et al., 2003) caffeic acid  and ferulic acid (Wahdan, 1998), and vanillic acid, cinnamic acid, and benzoic acid (Weston et al., 1999; Weston et al., 2000).

1.5.4.5. Antioxidant Activity

Antioxidants are substances that protect wound tissues from being damaged by oxygen radicals. The free radicals may be produced by hydrogen peroxide and cause cellular damage. Free radicals are involved in cell toxicity and can alter cell biomolecules such as proteins, carbohydrates, lipids and nucleic acids causing  cell death (Alvarez-Suarez et al., 2013).

Gheldof et al., (2002) analysed the antioxidant activity in different honey fractions and determined that most of the antioxidant components were found in the water-soluble fraction. These include gluconic acid, protein, ascorbic acid, hydroxymethylfuraldehyde, and the combined activities of the enzymes glucose  oxidase, catalase and peroxidase. The same study also showed that the phenolic compounds in honey contributed very significantly to its antioxidant capacity.

When honey is diluted the release of high levels of hydrogen  peroxide  may  lead to tissue damage by formation of free radicals such as hydroxyl and superoxide. Many honeys including manuka honey have the ability to quench free radicals. This property may play a role in reducing inflammation and chronic wound infection (Henriques et al., 2006)

A recent study was completed by Van de Berg et al., (2008) with regard to the antioxidant level in buckweat honey showing that this type of honey reduced the level  of reactive oxygen species (ROS) which affect the wound healing  process.  Also,  beside the low pH and high acidity buckwheat honey was shown to contain high amounts of phenolic components that aid the antimicrobial mechanisms and block the oxidative reaction system (Alvarez-Suarez et al., 2013). In addition, several reports demonstrated the relationship between the antioxidant and the colour of honey, where darker honey exhibited higher antioxidant  content (Bogdanov et  al.,  2004; Estevinho et al., 2008; Turkmen et al., 2006). It has been thought that non-hydrogen peroxide activity in manuka honey may be due to plant derived components such as flavonoids and phenolic compounds. Recently, two research groups have  reported  that  the  activity of Leptospermum honeys correlates with the presence of methyglyoxal (MG),  an alpha-oxoaldehyde that reacts with macromolecules such as DNA, RNA and  proteins (Adams et al., 2008). High amount of MG was present in some manuka honey which is equivalent to the non-peroxide activity.  MG was,  therefore,  known as a bioactive complex responsible for the antibacterial activity in manuka honey  (Adamset al., 2008).

Recently Atrott and Henle (2009) studied the presence of methylglyoxal in 61 samples of manuka honey. They found that the antibacterial activity ranged between 12.4% to 30.9% equivalent to phenol concentration.

More recently Kwakman et al., (2010) discovered an antibacterial bee peptide called bee defensin-1 in honey. To date this peptide has been isolated only from  a honey used in the production of revamil and it was confirmed that this protein exhibits most of the antibacterial activity.  The  exact mechanism of bee defensin-1 on bacteria  is not yet known.

1.5.7. Therapeutic Properties of Honey

Researchers have reported that honey is becoming acceptable  as a  reputable and effective therapeutic agent by Practitioners of conventional medicine and by the general public. Its beneficial role has been endorsed to its antimicrobial, anti- inflammatory and anti-oxidant activities as well as boosting of the immune system (Mohapatra et al., 2011; Sherlock et al., 2010; Buba et al., 2013; Fahim et al., 2014). 1.5.5.1.Antimicrobial Activity

The antimicrobial activity is very important therapeutically, especially in situation where the body’s immune response is insufficient to clear infection. In other words, it has shown powerful antimicrobial effects against pathogenic and non- pathogenic micro-organisms (yeasts and fungi) even against those that developed resistance to many antibiotics (Fahim et al., 2014). The antimicrobial effects could be bacteriostatic or bactericidal depending on the concentration that is used. However,  such activity has been attributed to certain factors like high osmolarity (low water activity), acidity (low pH), and hydrogen peroxide and nonperoxide components (Sherlock et al., 2010).

Furthermore, honey is a supersaturated sugar solution; these sugars have high affinity for water molecules leaving little or no water to support the growth of micro- organisms (bacteria and yeast). Consequently, the  micro-organisms  become  dehydrated and eventually die (Mohapatra et al., 2011). In addition, the natural acidity of honey will inhibit many pathogens.

According to Fahim et al. (2014), the usual pH range of most of the pathogens  is around 4.0- 4.5. However, the major antimicrobial activity has been found to be due

to hydrogen peroxide, produced by the oxidation of glucose by the enzyme glucose- oxidase, when honey is diluted. As hydrogen peroxide  decomposes,  it  generates  highly reactive free radicals that react and kill the bacteria. In most cases, the peroxide activity in honey can be destroyed easily by heat or the presence of catalase.

Notwithstanding, some honeys have antibacterial action separate  to  the peroxide effect, resulting in a much more persistent and stable antibacterial action (non-peroxide activity).They are however called  “non-peroxide  honeys.  Manuka honey (Leptospermum scoparium) from New Zealand and jelly bush (Leptospermum polygalifolium) from Australia are non-peroxide honeys which are postulated  to  possess unidentified active components in addition to the production of hydrogen peroxide. They retain their antimicrobial activity even in the presence  of  catalase (Buba et al., 2013).

Weston (2000) suggested that the main part of this activity might be of  honeybee origin, while part may be of plant origin. The compounds exhibiting this activity can be extracted with organic solvents (e.g. n-hexane,  diethyl  ether, chloroform, ethyl acetate) by liquid-liquid (Manyi-Loh et al., 2010) or solid phase extraction methods (Aljadi and Yusoff, 2003). The extracted compounds have been reported to include flavonoids, phenolic acids, volatile compounds (ascorbic acid, carotenoid-like substances, organic acids, neutral lipids, and Maillard  reaction products), amino acids and proteins (Vela et al., 2007).

Other important effects of honey have been linked to  its  oligosaccharides.  They have prebiotic effects, similar to that of fructo-oligosaccharides (Sanz et  al., 2005). The oligosaccharides have been reported to cause an increase in population of bifidobacteria and lactobacilli, which are responsible for maintaining a  healthy intestinal microflora in humans.As a matter of fact, Lactobacillus  spp.  protect  the body against infections like salmonellosis; and Bifidobacterium spp restrict the over- growth of the gut walls  by yeasts or bacterial pathogens and,  perhaps reduce the risk  of colon cancer by out-competing putrefactive bacteria capable of liberating  carcinogens (Kleerebezem and Vaughan, 2009).

The use of honey as a traditional remedy for microbial infections dates back to ancient times (Lusby et al., 2005). Research has been conducted on manuka (L. scoparium) honey (Visavadia et al., 2006), which has been demonstrated to  be  effective against several human pathogens, including Escherichia coli (E. coli), Enterobacter aerogenes, Salmonella typhimurium, S. aureus (Tan et al., 2009).

Laboratory studies have revealed that the honey is effective against methicillin-  resistant S. aureus (MRSA), streptococci and  vancomycin  resistant  Enterococci  (VRE) (Rajeswari et al., 2010).

However, the newly identified honeys may have  advantages  over  or similarities with manuka honey due to enhanced antimicrobial activity,  local  production (thus availability), and greater selectivity against medically important organisms (Tan et al., 2009). The coagulase negative staphylococci are very similar to

S. aureus (Rajeswari et al., 2010) in their susceptibility to honey of  similar  antibacterial potency and more susceptible than Pseudomonas aeruginosa and Enterococcus species (Fahim et al., 2014). The disc diffusion method is mainly a qualitative test for detecting the susceptibility of bacteria to antimicrobial substances; however, the minimum inhibitory concentration (MIC)  reflects  the  quantity  needed for bacterial inhibition. Following the in vitro methods, several bacteria (mostly multidrug resistant; MDR) causing human infections that were found susceptible to honeys (Mohapatra et al., 2011; Fahim et al., 2014).

Zone diameter of inhibition The zone diameter of inhibition (ZDI) of different honey samples (5%-20%)   has been determined against E. coli O157: H7 (12 mm -24 mm) and S. typhimurium (0 mm -20 mm) (Badawy et al., 2004). The ZDIs of Nigerian honeys were found to be (20-21) mm, (15-16) mm and (13-14) mm for S. aureus, P. aeruginosa and E. coli, respectively (Agbagwa and Frank- Peterside, 2010). Agbagwa and Frank- Peterside (2010) and Anyanwu (2012) examined different honey samples: Western Nigerian honey, Southern Nigerian honey, Eastern Nigerian honey and Northern  Nigerian  honey, and compared their abilities to inhibit the growth of S. aureus, P. aeruginosa,

E. coli and Proteus mirabilis (P. mirabilis) with an average of ZDIs (5.3-11.6) mm, (1.4-15.4) mm , (4.4-13.5) mm and (9.1-17) mm, respectively, and with honey concentrations of 80%-100%. The extracts of raw and processed honey showed ZDI (6.94-37.94) mm, against gram-positive bacteria viz., S. aureus, Bacillus subtilis, Bacillus cereus, as well as gram negative bacteria like E. coli, P. aeruginosa and S. enterica serovar Typhi (Chauhan et al., 2010).

Minimum inhibitory concentration The minimum inhibitory concentration(MIC) assay showed that a lower MIC was observed with ulmo (Eucryphia cordifolia) honey (3.1% - 6.3% v/v) than with manuka honey (12.5% v/v) for MRSA isolates; for the E. coli and Pseudomonas strains equivalent MICs were observed (12.5% v/v) (Sherlock et al., 2010). The MICs for Tualang honey ranged 8.75% - 25%, while those for manuka honey ranged 8.75% - 20% against many pathogenic gram-positive and gram negative bacteria (Tan et al., 2010). The MICs of manuka, heather, khadikraft and local honeys against clinical and environmental isolates of P. aeruginosa were recorded as 10% - 20%,  10% - 20%,  11% and 10% - 20%, respectively (Mullai and Menon, 2007). The MICs of  A.  mellifera honey ranged (126.23 - 185.70) mg/ml and of Tetragonisca angustula honey (142.87 - 214.33) mg/ml against S. aureus (Miorin et al., 2003). The Egyptian clover honey MIC was 100 mg/ml for S. typhimurium and E. coli O157:H7 (Badawy et al., 2004). The Nilgiri honey MICs were 25%, 35% and 40% for S. aureus, P. aeruginosa and E. coli, respectively (Rajeswari et al., 2010). MIC values of honey extracts were found in the range of (0.625-5.000) mg/ml, for S. aureus, B. subtilis, B. cereus, and gram-negative bacteria (E. coli, P. aeruginosa and S. typhi (Chauhan et al., 2010).

By visual inspection, the MICs of Tualang honey ranged 8.75% - 25%  compared with those of manuka honey (8.75% - 20%) against wound and enteric microorganisms: Streptococcus pyogenes, coagulase-negative Staphylococci, MRSA, Streptococcus agalactiae, S. aureus, Stenotrophomonas maltophilia (S. maltophilia), Acinetobacter baumannii, S. typhi, P. aeruginosa, Proteus mirabilis, Shigella flexneri,

E. coli , Enterobacter cloacae (Tan et al., 2010). Six bacterial strains  from  burn- wound patients, namely, Aeromonas schubertii, Haemophilius paraphrohaemlyticus, Micrococcus luteus, Cellulosimicrobium cellulans, Listonella anguillarum and A. baumannii had MICs of 35%-40%, 35%-40%, 35%-40%, 25%-30%, respectively, as has been reported by Hassanein et al. (2010). The honeys were inhibitory at dilutions down to 3.6% - 0.7 % (v/v), for the pasture honey, 3.4% - 0.5% (v/v), and for the manuka honey, against coagulase-negative Staphylococci (Fahim et al., 2014).

Time-kill study

The kill kinetics provides more  accurate description of antimicrobial activity    of antimicrobial agents than does the MIC (Tan et al., 2010). In our earlier study, we explored the time-kill activity of autoclaved honey against E. coli, P. aeruginosa and

S. Typhi in order to establish the potential efficacy of such local honey (not studied before) collected from villages (Mandal and  Mandal,  2011).  Antibiotic  susceptible and resistant isolates of S. aureus, S. epidermidis, Enterococcus faecium, E. coli, P. aeruginosa, E. cloacae, and Klebsiella oxytoca, etc, were killed within 24 h by 10%- 40% (v/v) honey (Kwakman et  al., 2011).  Thus, more studies are  required to establish various local honeys based upon kill kinetics and their effective in vivo application against MDR infections.