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A Comparative Study of Antibacterial and Antioxidant Activities of Wild Honey (Sunflower and Eucalyptus) and Commercial Honey

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Bacterial infections have been associated with numerous diseases that pose threat to public health. Eradication of these infectious pathogens is a global challenge due to prevailing of multidrug resistance phenomenon. Therefore, there is unmet need to find new antimicrobial agents from natural sources such as honey. Well diffusion and Minimum inhibitory concentrations assays were used to assess the antibacterial activity of two locally wild honeys viz., Eucalyptus, Sunflower and a commercial honey (Qurshi Honey Gold) against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, and Serratia marcesnces. Thin layer chromatography and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay were used to evaluate the bioactive constituents and antioxidant agents, respectively. Maximum inhibition zones (31.6 ± 2.88 mm, 36 ± 0.00 mm, and 30.00 ± 2.64 mm) with all honey samples at 100% concentration attained against K. pneumoniae while S. marsences showed inhibition zones (38.3 ± 2.8 mm, 35.33 ± 3.78 mm, and 31.68 ± 2.57 mm) at the same concentration. Eucalyptus and sunflower honeys exhibited 24.33 ± 4.04 mm, 36.3 ± 1.5 mm, 32.00 ± 1.00 mm, and 27.2 ± 1.53 mm against S. aureus and P. aeruginosa respectively. Activity index reported the effect use of honey against clinical bacterial pathogens when compared with antibiotics. Eucalyptus honey showed significant antioxidant activity at 100% concentration. The appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts and acetone solvent system indicated the presence of alkaloids, polyphenols, flavonoids, and steroids. All data were analyzed statistically using Two-way ANOVA and level of significance were recorded at P
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Article
Journal of
Pharmaceutical Sciences
and Pharmacology
Vol. 2, 1–8, 2014
www.aspbs.com/jpsp
A Comparative Study of Antibacterial and Antioxidant
Activities of Wild Honey (Sunflower and Eucalyptus)
and Commercial Honey
Uzma Azeem Awan, Shaukat Ali, and Saiqa Andleeb
Microbial Biotechnology laboratory, Department of Zoology, University of Azad Jammu and Kashmir, Muzzafarabad, Pakistan
Bacterial infections have been associated with numerous diseases that pose threat to public health. Eradication of these
infectious pathogens is a global challenge due to prevailing of multidrug resistance phenomenon. Therefore, there is
unmet need to find new antimicrobial agents from natural sources such as honey. Well diffusion and Minimum inhibitory
concentrations assays were used to assess the antibacterial activity of two locally wild honeys viz., Eucalyptus, Sun-
flower and a commercial honey (Qurshi Honey Gold) against Escherichia coli, Pseudomonas aeruginosa, Klebsiella
pneumonia, Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, and Serratia marcesnces.
Thin layer chromatography and 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay were used to evaluate the
bioactive constituents and antioxidant agents, respectively. Maximum inhibition zones (316±288 mm, 36±000 mm,
and 3000 ±264 mm) with all honey samples at 100% concentration attained against K. pneumoniae while S. marsences
showed inhibition zones (383±28 mm, 3533 ±378 mm, and 3168 ±257 mm) at the same concentration. Eucalyp-
tus and sunflower honeys exhibited 2433 ±404 mm, 363±15 mm, 3200 ±100 mm, and 272±153 mm against S.
aureus and P. aeruginosa respectively. Activity index reported the effect use of honey against clinical bacterial pathogens
when compared with antibiotics. Eucalyptus honey showed significant antioxidant activity at 100% concentration. The
appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts and
acetone solvent system indicated the presence of alkaloids, polyphenols, flavonoids, and steroids. All data were analyzed
statistically using Two-way ANOVA and level of significance were recorded at P<005. It was concluded that honey could
be used as cost-effective source of bioactive compounds that has potential to treat bacterial pathogenic infections.
KEYWORDS: Honey, Thin Layer Chromatography, Antioxidant Activity, Antibacterial Activity, Minimum Inhibitory Concentrations of
Honey.
INTRODUCTION
Antibacterial strength of honey has been attributed due to
its strong osmotic effect, naturally low pH, and phytochem-
ical factors (Taormina et al., 2001). Hydrogen peroxide
produced by honey has a vital role in the antimicrobial
activity (Kaˇ
cániová et al., 2011). Traditionally, honey has
been reported as valuable source of energy having both
antioxidant and antimicrobial characteristics. It is a con-
centrated aqueous solution of carbohydrates, organic acids,
amino acids, aromatic substances, minerals, pigments,
pollen grains and waxes (Sanz et al., 2004; Maddocks and
Author to whom correspondence should be addressed.
Email: drsaiqa@gmail.com
Received: 28 March 2014
Accepted: 17 July 2014
Jenkins, 2013). Previous study confirmed the antimicrobial
activity of honey against a wide range of microbes like
multidrug resistant pathogens (Maeda et al., 2008). Some
studies verified the antimicrobial action of honey against
Campylobacter jejuni, Escherichia coli, Shigella dysente-
riae, and Salmonella enterocolitis (Voidarou et al., 2011),
Mycobacterium (Asadi et al., 2003) Staphylococcus aureus,
Enterococci (Lin et al., 2011) and the development of
Streptococcus pyogenes biofilms (Maddocks et al., 2012).
The anti-candida activity of the honey has also been
reported (Ahmed et al., 2012). It has been observed that
the component of honey like methylglyoxal (MG) used as a
precursor for the synthesis of advanced glycation end prod-
ucts (Stephens et al., 2010; Majtan et al., 2014). Honey has
anti-oxidant, anti-bacterial and anti-inflammatory proper-
ties (Reza et al., 2013; Montenegro and Mejías, 2013).
J. Pharm. Sci. Pharmacol. 2014, Vol. 2, No. 1 2333-3715/2014/2/001/008 doi:10.1166/jpsp.2014.1020 1
A Comparative Study of Antibacterial and Antioxidant Activities Awan et al.
Now a day, there is a growing demand for natural
products and bio-organics in the human diet due to the
negative effects of synthetic food additives on human
health. Honey serves as a source of natural antioxidants,
which are effective in reducing the risk of cancer, heart
disease, infected and chronic wounds, immune system
decline, the autism disease, asthma, gastrointestinal disor-
ders, skin ulcers, and cataracts (Dastouri et al., 2008). The
current research was conducted to evaluate antibacterial
and antioxidant activities of honey samples from different
localities of Azad Jammu and Kashmir, Pakistan. It was
hypothesized that certain concentrations may display good
inhibitory effects against pathogenic bacteria and therefore
may aid in the development of antimicrobials and antiox-
idant supplements.
EXPERIMENTAL WORK
Samples Collections
All honey samples were collected directly from the combs,
stored in fine plastic containers and labeled with names,
numbers and date of collection. Sunflower and Eucalyp-
tus honeys were collected from Kail (Neelum Valley) and
Leepa (Jhelum valley), Muzaffarabad, Azad Jammu and
Kashmir, Pakistan. The commercial honey (Qarshi Honey
Gold) was purchased from local market of Muzaffarabad,
Azad Jammu and Kashmir, Pakistan. Four species of hon-
eybees are found in Northern areas and Azad Jammu and
Kashmir, Pakistan. Three species are indigenous (Apis dor-
sata,Apis cerana,andApis florae) and one exotic species
(Apis mellifera).
Sample Preparation
Unnecessary material such as sticks, dead bees, wax and
particles of combs were removed by straining through
cheese cloth. Different concentrations of both wild and
commercial honeys viz., 5%, 10%, 15%, 20% and 50%
prepared with double distilled water (d2H2O) through
serial dilutions of 100% honey. These dilutions were used
for the estimation of minimum inhibitory activity (MICs).
The least concentration that did not show growth of test
organisms was considered as the MICs. The concentrations
were stored at 4 C for further processes.
Determination of Antioxidant Potential
To evaluate the antioxidant potential of honeys, the chem-
ical reagent ABTS {2,2-azinobis-(3-ethylbenzothiazoline-
6-sulfonic acid} was used according to the method
described of Re et al., (1999). The ABTS+stock solu-
tion was prepared by reacting potassium persulphate
(2.45 mM) and ABTS+(7 mM), then the mixture was
kept for 16 h to generate ABTS+free radicals and their
absorbance were recorded at 734 nm (AoControl . For tests,
1 ml of ABTS and running solution were mixed with
20–200 g/ml of different compounds. Water is used in
case of control sample. The absorbance of test samples
(AiSamplewas also observed at 734 nm. The percentage
radical scavenging activity (% RSC) was calculated using
the formula: %RSC =[(AoControl AiSample /AoControl×
100%.
Agar Well Diffusion Method
Bacterial pathogens viz., S. aureus, S. pyogene, S. epi-
dermidis, P. aeruginosa, K. pneumonia,E. coli and
S. marcescens were isolated and identified by Awan et al.,
(2013). The antibacterial activity of different concentra-
tions of honey samples was tested by agar well diffusion
method (Dastouri et al., 2008) Nutrient agar and Nutri-
ent Broth Media (NAM; Oxide CMOO3 and NBM; CM1)
were used for bacterial culture. The overnight prepared
culture was mixed with freshly prepared nutrient agar
medium (NAM) at 45 C and was poured into the sterilized
Petri dishes. All Petri dishes were kept at room tempera-
ture in laminar flow for solidification. After solidification,
three wells were made in each agar plate with a flamed,
cooled cylinder of 5 mm diameter and sterile needle was
used for the removal of agar plugs. The volume (50 l)
of the various concentrations of EH, OH and CH was sep-
arately dispensed into each well of prepared plates. Dou-
ble distilled water was used as negative control. Before
each experiment, the optimal density (OD) of bacterial
growth 107 colony forming units (cfu)/ml was measured
through spectrophotometer at 600 nm (Seeley et al., 2001).
The inhibitory effect was recorded in millimeter (mm)
by measuring the diameter of the zone of growth inhibi-
tion after 24–48 h. The growth inhibition was recorded as
(0) for no sensitivity, (1–10) for low sensitivity, (11–20)
for moderate, and (21-above) for high sensitivity. Antibi-
ogram of various groups of standard antibiotics such as
aminoglycosides (Tobramycin 5 g/ml), Penicillins (Peni-
cillin G 10 g/ml), Tetracyclines (Tetracycline 10 g/ml),
Sulfonamides (Sulphamethoxyzole 10 g/ml) against test
pathogens was also assessed through agar disc diffusion
method and used as positive control (Prescott et al., 1999).
Chemical Screening Through Thin
Layer Chromatography
The indication of major phytochemical was evaluated by
thin layer chromatography (TLC) using precoated Silica
gel 60F264 plates (Wagner and Bladt, 2004). For TLC
studies, chloroform extract of honey was used. Crude
honey (100 g) of pure honey was diluted with 150 ml
of d2H2O and extracted by using 150 ml of chloroform.
After mixing, the honey extract was concentrated on a
rotary evaporator at 50 C. Different screening systems
were used to get better resolution of components. The
developed plates were observed using various reagents
(anisaldehyde/H2SO4and UV light (254–336 nm). Reten-
tion factor (Rfvalue of each spot was calculated
2J. Pharm. Sci. Pharmacol. 2, 1–8, 2014
Awan et al. A Comparative Study of Antibacterial and Antioxidant Activities
in centimeter (cm) as Rf=distance travelled by the
solute/distance travelled by the solvent.
STATISTICAL ANALYSIS
Each treatment and experiment was replicated 3 times
and data has been expressed as Mean ±SD. Statistical
analyses were performed using GraphPad Prism 6 (Win-
dows version 6.04). Two-way ANOVA and activity Index
was used to compare the values of samples with posi-
tive control. A P value <005 was regarded as indicating
significant differences and levels of significance are indi-
cated as ∗∗∗∗(highest significance), ∗∗∗ (highly significance),
∗∗(moderate significance), and (Low significance).
RESULTS/FINDINGS
Antioxidant Activity
Concentration dependant antioxidant activity of three
honeys was found. Depending upon their various
concentrations the scavenging potential values of three
honey samples were higher in EH (95 ±211%), moderate
in SH (79 ±218%), and comparatively low in CH (67 ±
058%) see Figure 1. Results indicated a notable effect of
all tested honey samples on ABTS+radical. We found that
honey possessed rich properties of antioxidants and could
be used as an antioxidant agent.
Antibacterial Activity
In current research the sensitivity of bacterial pathogens
were observed against all the concentrations of honey
except 5% w/v concentration, which showed no signif-
icant inhibition by agar well diffusion method (Fig. 2).
There was no evidence of inhibition of P. aeruginosa
and E. coli with concentrations up to 20% w/v, how-
ever it was found that EH, CH, and SH honey samples
still had some antibacterial activities at that concentration.
From current research it was observed that two of the
tested bacteria such as K. pneumonia and S. marcescens
were most sensitive to all honey samples at 100%
concentration and showed the maximum growth inhibi-
tion (316±288 mm, 360±000 mm, 3000 ±246 mm,
Figure 1. Antioxidant Activity of MICs of Honey. EH (Eucalyp-
tus Honey), SH (Sunflower honey), CH (Commercial honey),
MICs of Honey are represented as (5, 10, 15, 20, 50 and 100%).
3533 ±378 mm, 383±28 mm, and 36 ±100 mm).
Commercial honey also exhibited a potent antibacterial
activity against K. pneumonia, S. epidermidis and S.
marcescens (3000 ±264 mm, 3433 ±152 mm, and
3168 ±257 mm) while the eucalyptus honey caused
greater inhibition of P. aeruginosa (3200 ±100 mm), S.
aureus (2433 ±404 mm), and E. coli (2833 ±288 mm)
at the same concentration. Similarly, S. pyogenes, S.
aureus,andS. marcessens were significantly inhibited by
sunflower honey at 100% concentration (2100 ±17 mm,
2400 ±000 mm, and 3110 ±033 mm). On the other
hand negative control had no effect on all tested clinical
bacterial pathogens.
Antibiogram and Activity Index Analysis
The four different classes of standard antibiotics were used
to test the sensitivity of bacterial pathogens called antibi-
ogram analysis (Table I). It was found that sulfamethoxy-
zol and penicillin G had no effect on the growth of E.
coli, S. aureus and S. pyogenes. Similarly, tetracycline had
no effect on S. pyogenes,S. aureus,andS. marcesse-
cens. K. pneumoniae and S. epidermidis showed resistance
against penicillin G. On the other hand, maximum zone of
inhibition was recorded against K. pneumoniae and S. epi-
dermisdis in the presence of sulfamethoxyzole (31±0 mm,
and 33 ±0 mm) and tobramycin (20 ±0 mm and 30 ±
0 mm). Through activity index analysis it was recorded
that eucalyptus honey at 100% concentration showed sig-
nificant results when compared with all tested positive
controls. Similarly, Sunflower honey showed moderate sig-
nificant results at 20%∗∗, highly significant at 50%∗∗∗ and
highest significant at 100%∗∗∗∗ concentration as indicated
in Table I. On the other hand, commercial honey had also
maximum effect against all tested pathogens at 20%, 50%,
and 100% when compared with the antibiotics. In case
of negative control, activity index indicated the significant
effect of all tested honey samples. It was concluded that
the level of significance of zone of inhibition of tested
honey at P<005 was sunflower honey >Eucalyptus
honey >commercial honey (SH >EH >CH).
Thin Layer Chromatography
In chemical screening, performed by TLC on the crude
extracts of honey, an impressive diversity of the chemical
constituents can be seen. In TLC analysis, different absorb-
ing bands were observed under short and long wavelength
of UV i.e., 254 nm and 336 nm, respectively. Prominent
colored bands of different crude extracts were observed
by staining with anisaldehyde/H2SO4and also observed
under UV. The colored bands like red, orange, brownish to
yellow, red to brown were indicating the presence of dif-
ferent types of functional groups like amines, phenols etc.
EH, SH and CH samples showed significant colored bands
on TLC plates in chloroform/methanol, ethyl acetate/acetic
acid and n-hexane/ethyl acetate while slightly moderate
separation was seen in n-hexane/acetone system (Fig. 3).
J. Pharm. Sci. Pharmacol. 2, 1–8, 2014 3
A Comparative Study of Antibacterial and Antioxidant Activities Awan et al.
Figure 2. Zone of inhibition of human pathogenic bacteria by MICs of honey through agar well diffusion method. Levels of
significance at P<005 are indicated as ∗∗∗∗ (highest significance), ∗∗∗ (highly significance), ∗∗ (moderate significance) and (low
significance). EH (Eucalyptus Honey), SH (Sunflower honey), CH (Commercial honey), MICs of Honey are represented as (5, 10,
15, 20, 50 and 100%).
4J. Pharm. Sci. Pharmacol. 2, 1–8, 2014
Awan et al. A Comparative Study of Antibacterial and Antioxidant Activities
Tab le I . Activity index (AI) analysis of antibiotics and MICs of different honeys.
Eucalyptus honey Sunflower honey Commercial honey
Bacterial Antibiotics (Zone of
pathogens 5% 10% 15% 20% 50% 100%∗∗∗∗ 5% 10% 15% 20%∗∗ 50%∗∗∗ 100%∗∗∗∗ 5% 10% 15% 20%∗∗ 50%∗∗∗ 100%∗∗∗∗ inhibition mm)
Klebsiella 0.02 0.11 0.47 0.58 0.69 1.02 A>H 0.35 0.48 0.75 0.93 1.16 A >H 0.36 0.54 0.69 0.84 0.91 Sulfamethoxyzol (31 ±0)
pneumoniae H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>APenicillin G (0 ±0)
A>HA>H 1.04 1.28 1.54 2.26 A>H 0.86 1.07 1.57 2.07 2.57 A>H 0.85 1.28 1.64 2.00 2.14 Tetracycline (14 ±0
A>HA>H 0.73 0.9 1.08 1.58 A>H 0.55 0.75 1.10 1.45 1.80 A>H 0.6 0.90 1.15 1.40 1.50 Tobramycin (20 ±0)
Streptococcus H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>ASulfamethoxyzol (0 ±0)
pyogenes H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>APenicillin G (0 ±0)
H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>ATetracycline (0 ±0)
A>HA>HA>HA>HA>H3.00 A>HA>H1.60 3.80 4.20 4.80 A>H2.4 3.60 4.60 5.60 6.00 Tobramycin (5 ±0)
Staphylococcus A>HA>HA>H 0.42 0.622 0.66 A >HA>HA>HA>H 0.51 0.57 A >H 0.36 0.37 0.54 0.90 1.03 Sulfamethoxyzol (33 ±0)
epidermidis H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>APenicillin G (0 ±0)
A>HA>HA
>H 1.30 1.93 2.03 A>HA>H1.10 1.30 1.70 1.90 0.9 1.2 1.30 1.80 3.00 3.40 Tetracycline (10 ±0)
A>HA>HA>H 0.43 0.64 0.67 A >HA>H 0.36 0.43 0.56 0.63 0.3 0.4 0.43 0.6 1.00 1.13 Tobramycin (30 ±0)
Staphylococcus H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>ASulfamethoxyzol (0 ±0)
aureus H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>APenicillin G (0 ±0)
H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>ATetracycline (0 ±0)
A>HA>HA>HA>H2.11 2.70 A>HA>HA>H 1.66 2.66 4.00 A>HA>HA>HA>H1.11 2.11 Tobramycin (9 ±0)
Serratia A>HA>HA>HA>HA>H4.42 1.62 1.75 2.50 3.5 3.87 4.75 0.22 A >H 0.22 0.50 A >H 0.30 Sulfamethoxyzol (8 ±0)
marcesscens A>HA>HA>HA>HA>H4.42 1.62 1.75 2.50 3.5 3.87 4.75 0.22 A >H 0.22 0.50 A >H 0.30 Penicillin G (8 ±0)
H>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>AH>ATetracycline (0 ±0)
A>HA>HA>HA>HA>H3.53 1.30 1.40 2.00 2.80 3.10 3.80 0.20 A >H 0.2 0.40 A >H 0.30 Tobramycin (10 ±0)
Pseudomonas A>HA>HA>HA>HA>H5.33 A>HA
>HA>HH=A 2.50 4.50 A>HA>H 0.83 1.16 1.83 2.83 Sulfamethoxyzol (6 ±0)
aeruginosa A>HA>HA>HA>HA>H5.33 A>HA>HA>HH=A 2.50 4.50 A>HA>H 0.83 1.16 1.83 2.83 Penicillin G (6 ±0)
A>HA>HA>HA>HA>H5.33 A>HA>HA>HH=A 2.50 4.50 A>HA>H 0.83 1.16 1.83 2.83 Tetracycline (6 ±0)
A>HA>HA>HA>HA>H2.46 A>HA>HA>HA>H1.15 2.07 A>HA>H 0.38 0.53 0.84 1.30 Tobramycin (13 ±0)
Escherichia H>AH>AH>AH>AH>AH>A0 0 0H>AH>AH>A0 0H>A0 H>AH>ASulfamethoxyzol (0 ±0)
coli H>AH>AH>AH>AH>AH>A0 0 0H>AH>AH>A0 0H>A0 H>AH>APenicillin G (0 ±0)
H=AH=A2.00 7.50 11.00 14.0 A>HA>HA>H4.50 6.50 7.50 A>HA>H1.00A>H 1.00 5.00 Tetracycline (2 ±0)
H=AH=A2.00 7.50 11.00 14.0 A>HA
>HA>H4.50 6.50 7.50 A>HA>H1.00A>H 1.00 5.00 Tobramycin (2 ±0)
Notes:H>A and >1 value of AI indicates MICs of honey has higher effect against bacterial pathogens compared to antibiotics; A >Hand<1 value of AI indicates antibiotics has higher effect against bacterial
pathogens compared to MICs of honey. H =A and 1 value of AI indicates both extract and antibiotic have equal effect whereas 0 indicates both extract and antibiotic has no effect. Levels of significance at P<005
are indicated as ∗∗∗∗(highest significance), ∗∗∗ (highly significance), ∗∗(moderate significance) and (low significance).
J. Pharm. Sci. Pharmacol. 2, 1–8, 2014 5
A Comparative Study of Antibacterial and Antioxidant Activities Awan et al.
Figure 3. Thin layer chromatography of honey extracts by using various solvent systems. S1, S2, S3 indicates the presence of
spots on TLC developed plates of honey samples. EH (Eucalyptus Honey), SH (Sunflower honey), CH (Commercial honey).
DISCUSSION
Antioxidant Activity
Pharmacological effects of most natural products used for
medicinal purposes have been correlated to their posses-
sion of antioxidant activity (Sofidiya et al., 2006). Basi-
cally antioxidant used to reduce the oxidative reactions
within the human body, mainly due to their redox prop-
erties, which play significant role in neutralizing and
adsorbing decomposing peroxides or free radicals (Louli
et al., 2004). The antioxidant activity of honey has been
attributed due to the presence of phenolic, flavonoids, cat-
alytic action of catalase and glucose oxidase (Djeridane
et al., 2006). Honey is reported to scavenge free radi-
cals effectively. Honey might be a novel antioxidant in
the management of chronic diseases frequently linked with
oxidative stress (Omotayo et al., 2012).
Pharmacological Analysis of Honey
Adebolu, (2005) reported the antimicrobial action of honey
but most of the reports are conflicting and are not substan-
tiated by enough scientific proof. Thus, the present study
is in confirmation with previous reports of various litera-
tures (Obaseiki-Ebror et al., 1983). As far as the organisms
are concerned, the present report conformed the findings
of Obaseiki–Ebror et al., (1983) and suggested that three
major systems i.e., inhibines, high osmotic pressure and
acidity are responsible for the antimicrobial activity of
honey (Hamid and Saeed, 1991). Staphylococcus aureus
has always been employed in many microbiological eval-
uations of honey because of its high sensitivity (Cooper,
1999) as consistent with our findings but the reason for
this unusual sensitivity is still unknown. It may be related
to the sensitivity of S. aureus to acidic environment of
honey (Molan, 2002). The outcomes of current research
showed that the growth of both Gram positive and negative
bacteria was equally inhibited by MICs of honey samples.
These results did not agree with the reported results by
Mohapatra et al., (2011). He showed that the Gram nega-
tive bacteria are more susceptible to the inhibitory action
of honey than Gram-positive bacteria.
Chauhan et al., (2010) illustrated that E. coli and P.
aeruginosa were the most susceptible bacteria to honey.
Current study showed variability in antibacterial activity
of honey samples. This variation has also been reported by
Taormina et al., (2001). Significant results were observed
by honey at low MIC values when four different concen-
trations of wild and commercial honey were tested against
E. coli and P. aeruginosa as reported by Wilkinson and
Cavanagh, (2005). These findings are quite compatible
with our results where EH showed strong growth inhibi-
tion of P. aeruginosa, S. aureus,andE. coli. According to
our results all tested honey samples showed high inhibitory
zone of K. pneumonia and S. marcesscens. According to
previous reports, the inhibitory effect of EH and SH honey
produced by the honeybee (Apis mellifera) was highest
inhibition of E. coli followed by S. aureus at all concen-
trations, except at highly diluted samples Whereas less
6J. Pharm. Sci. Pharmacol. 2, 1–8, 2014
Awan et al. A Comparative Study of Antibacterial and Antioxidant Activities
antibacterial activity against P. aeruginosa was recorded
(Osho and Bello, 2010). Our results showed similarity with
the work of Vica et al., (2014). According to their stud-
ies the most sensitive to the antibacterial activity were the
two Staphylococus strains (the largest diameter of inhibi-
tion zone was 18 mm) and B. subtilis strains (13.5 mm).
The strains of B. cereus,E. coli,L. monocytogenes and
Salmonella spp. were found to present resistance to some
of the honey samples. Manna, sunflower and polyfloral
honeys presented significant antibacterial activity while
acacia and linden honeys had a lower activity in terms
of the number of sensible strains (Vica et al., 2014).
Our results showed equal inhibition of Gram-positive and
Gram-negative bacteria but according to the Zahoor and
his coworkers, all the tested honeys (Acacia modesta,
Prunus persica,Zizyphus sativa and Isodon rogosus)were
more active against Gram-negative bacterial strains than
the Gram-positive bacterial strains (Zahoor et al., 2014).
Thin Layer Chromatography
The results revealed that methanolic solvent system is
much better for separation of components than less polar
or non polar systems. However, ethanol/water or ace-
tone/water were better solvents compared to ethanol or
acetone for the separation of various components (Yilmaz
et al., 2006). Our results had reasonable similarities with
some earlier reports stating that non polar compounds like
hydrocarbons, fatty acids and waxes present in natural
products could be extracted with chloroform/methanol sol-
vent system (Stalikas, 2007). When we discussed about the
different spot colors, we found interesting literature about
the color tests for identification of different colors of var-
ious compounds. As in the present study, the chloroform
extracts of three honeys showed pink to yellow band when
stained with anisaldehyde/sulphuric acid. It was reported
in the literature that these specific spots indicated the pres-
ence of phenols in the extracts being studied (Stahl, 1969).
On the other hand, purple color spots on staining with
the same reagents in the present study indicated the pres-
ence of hydroquinone and other hydroxyphenylpropane
derivatives, brownish color indicating the presence of ter-
penes and pink to brown and yellow color under the UV
light (365 nm) indicated the presence of alkaloids (Stahl,
1969). The components of honey can be successfully sep-
arated using TLC method. Various types of compounds
were identified by comparing the Rf-values (obtained) with
the literature values (Kikuzaki et al., 1999). Polyphenolic
compounds in honey were determined by TLC in some
published studies (Aljadi and Kamaruddin, 2004). Thin
layer chromatography has also been employed for the
detection of flavonoids in honey samples (Berahia et al.,
1993). Our results had close resemblance with the previous
findings (Cabras et al., 1999). The result of present study
was also in conformity with the work of Nwodo et al.,
(2010) who equally showed the significant separation of
honey by TLC.
CONCLUSIONS
The fact that inhibition of bacterial pathogens by these hon-
eys was superior, over the most common antibiotics used to
treat bacteria, makes them a novel source of antimicrobial
agents to treat drug resistant bacteria. Honey as a cheap
antibacterial agent can be used to manage chronic wound
types, such as burns, leg ulcers or surgical wounds. Usually
these wounds become infected with methicillin-resistant
Staphylococcus aureus or Pseudomonas.However,further
studies are needed to determine the volatile and non volatile
active ingredients in EH and SH as well as toxicological
testing. Hence, we conclude that honey can be effectively
used as antimicrobial and antioxidant agent to overcome
the problem of bacterial infections and multidrug resistant
microbial strains, as to enable and enhance the market rev-
enue throughout the world.
Conflict of Interest
The authors declare that they have no competing interests.
Authors’ Contributions
Uzma Azeem Awan carried out the honey concentrations,
antibacterial, antioxidant and TLC experiments. Shaukat
Ali analyzed the data and improved the manuscript. Saiqa
Andleeb supervised the research work protocols and pre-
pared the manuscript. All authors read and approved the
final manuscript.
Acknowledgments: The authors are grateful to Mr.
Muhammad Siddique Awan, Associate Professor, Depart-
ment of Zoology, University of Azad Jammu and Kashmir
for providing honey samples.
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A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.