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ANALYSIS OF MEDICINAL AND CHEMICAL PROPERTIES OF HONEYS
COLLECTED FROM SOME SELECTED LOCATIONS OF PAKISTAN
By
NADIA NOOR
M. PHIL. (UAF)
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSPHY
IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY & BIOCHEMISTRY
FACULTY OF SCIENCES
UNIVERSITY OF AGRICULTURE,
FAISALABAD, PAKISTAN
2015
iii
ACKNOWLEDGEMENT
I bow my head before Almighty Allah, the omnipotent, the omnipresent, the merciful,
the most gracious, the compassionate, the beneficent, who is the entire and only source of every
knowledge and wisdom endowed to mankind and who blessed me with the ability to do this
work. It is the blessing of Almighty Allah and His Prophet Hazrat Mohammad (Peace be
upon him) which enabled me to achieve this goal. .
I would like to take this opportunity to convey my cordial gratitude and appreciation to
my worthy, reverently and zealot supervisor Dr. Raja Adil Sarfraz, Assistant Professor,
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan. I
am really indebted to him for his accommodative attitude, patience and sympathetic behavior. I
would like to pay my deepest gratitude and appreciation to one of the member of my supervisory
committee Dr. Shaukat Ali, Assistant Professor, Department of Chemistry and Biochemistry,
University of Agriculture, Faisalabad, Pakistan. I am also extremely grateful to Dr. Muhammad
Shahid, (member of my supervisory committee), Associate Professor, Department of Chemistry
and Biochemistry, University of Agriculture, Faisalabad, Pakistan for his generous cooperation
and providing valuable suggestions during accomplishment of my Ph.D studies. I am deeply and
strongly obliged to Dr. Azam Tasneem, Chief Scientist, Head, Isotope Application Division,
Pakistan Institute of Nuclear Science & Technology, P. O. Nilore, Islamabad, Pakistan, for
assistance in the analysis on GCMS. I am thankful to Dr. Amer Jamil, Professor, Department of
Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan for his valuable
suggestions throughout of my Ph. D. studies. I pay my special thanks to all of my teachers who
taught me during my academic career. I am also thankful to the Central Hi-Tech Laboratory,
University of Agriculture, Faisalabad, Pakistan for technical support to complete my research
work. I am also very much grateful to Higher Education Commission (HEC), Pakistan, for
awarding me the Indigenous PhD Fellowship. I am also thankful to all my friends for
encouragement and their prayers for me.
Last but not least, I really acknowledge and offer my heartiest gratitude to all members of
my family my father and mother for patience, tolerance and prayers of my health and success
which enabled me to achieve this excellent achievement. I am also thankful to mamu (Ahsan
Ali) for moral support in every objective of my life. I also want to acknowledge my brothers
(Imran Nizami and Ali Asghar), bhabi and sister for encouragement and prayers of my health
and success which enabled me to achieve this excellent achievement.
Nadia Noor
D/O M. Ashraf Nizami
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TABLE OF CONTENTS
Chapter No. Title Page No.
Chapter 1 Introduction 1
Chapter 2 Review of literature 6
2.1 Species of honey bee 9
2.2 Physicochemical characterization 9
2.2.1 Ash and electrical conductivity (EC) 11
2.2.2 Color of honey 11
2.2.3 pH 13
2.2.4 Moisture contents 13
2.2.5 Proteins and free amino acid 13
2.2.6 Hydroxy methylfurfuraldehyde (HMF) 14
2.2.7 Sugar profile 15
2.2.8 Isotopic ratio analysis for detection of adulteration 16
2.2.9 Metal analysis 17
2.3 Bioactive composition of honey 19
2.3.1 Phenolics 19
2.3.2 Flavonoids 20
2.4 Medicinal properties of honey 22
2.4.1 Antioxidant potential 23
2.4.2 Antimicrobial properties of honeys 26
2.4.3 Wound healing properties 29
2.4.4 Antitumor potential 31
Chapter 3 Materials and Methods 34
3.1 Instruments used 34
3.2 Materials 35
3.3 Standards 35
3.4 Sampling 35
3.5 Physicochemical analysis 37
3.5.1 Physical analysis 37
3.5.2 Total protein contents 37
3.5.3 Total free amino acid 38
3.5.4 Total proline 38
3.5.5 Sugar analysis by HPLC 38
3.5.6 Mineral analysis 39
3.6 Isotopic ratio analysis of C12
/C13
41
3.6.1 Procedure for preparation of CO2 from honey samples 41
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Chapter No. Title Page No.
3.6.2 Description of the mass spectrometer analysis procedure 41
3.7 Bioactive contents 42
3.7.1 Total phenolic content (TPC) 42
3.7.2 Total flavonoids contents (TFC) 42
3.7.3 Total antioxidant contents (TAC) 42
3.7.4 Vitamin C. contents 43
3.7.5 Total carotenoid (TCC) and lycopene contents (TLC) 43
3.8 Antioxidant activities 44
3.8.1 DPPH radical scavenging activity (%RSA) 44
3.8.2 Ferric reducing antioxidant power assay 44
3.8.3 Peroxide scavenging activity 44
3.9 Antimicrobial activities 45
3.9.1 Antibacterial activity 45
3.9.1.1 Bacterial strains 45
3.9.1.2 Preparation of fresh bacterial culture 45
3.9.1.3 Plate assay/disc diffusion method 45
3.9.1.4 MIC against bacterial strains 45
3.9.2 Antifungal activity 45
3.9.2.1 Fungal strains 46
3.9.2.2 Preparation of fresh fungal culture 46
3.9.2.3 Plate assay 46
3.9.2.4 MIC against fungal strains 47
3.10 Potato disc assay 47
3.10.1 Preparation of honey solutions 47
3.10.2 Antitumor activities 47
3.11 Statistical analysis 48
Chapter 4 Results and Discussion 49
4.1 Physicochemical properties of honeys 49
4.1.1 Electrical conductivity (EC) 50
4.1.2 pH 52
4.1.3 Ash contents 55
4.1.4 Color of honey samples 57
4.1.5 Moisture contents 58
4.1.6 Refractive index 61
4.1.7 Total protein 61
4.1.8 Total free amino acids and proline content 63
4.1.9 Sugar profile 67
4.10 Mineral profile 75
4.10.1 Calcium 75
4.10.2 Magnesium 77
4.10.3 Iron 77
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Chapter No. Title Page No.
4.10.4 Zinc 78
4.10.5 Manganese 79
4.11 Bioindication of heavy metal toxicity of environment 81
4.11.1 Copper 81
4.11.2 Lead 82
4.11.3 Cobalt 83
4.11.4 Cadmium 86
4.11.5 Cromium 90
4.11.6 Nickle 90
4.12 Isotopic ratio analysis of C12
/C13
for detection of adulteration 91
4.13 Statistical analysis 92
4.13.1 Principal component analysis 92
4.13.2 Cluster analysis 95
4.2 Bioactive components of honeys 96
4.2.1 Total phenolic contents 96
4.2.2 Total flavonoid contents 99
4.2.3 Vitamin C. contents 103
4.2.4 β-carotene 105
4.2.5 Lycopene contents 108
4.3 Antioxidant profile 109
4.3.1 Total antioxidant contents 109
4.3.2 Free radical scavenging activities 112
4.3.2.1 DPPH scavenging activity 112
4.3.2.2 Ferric reducing antioxidant power 115
4.3.2.3 Peroxide scavenging activity 116
4.4 Antimicrobial activities 119
4.4.1 Antibacterial activities 119
4.4.2 Minimum inhibitory concentrations 124
4.4.3 Antifungal activity 128
4.5 Antitumor activity of Pakistani honeys 128
Chapter 5 Summary 132
Future Prospectives 134
Literature Cited 136
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LIST OF TABLES
Table No. Title Page No
2.1 Basic composition of honey for authenticity of honey 8
2.2 Relationship of honey color grade and Pfund scale 12
2.3 Sugar profile of different types of honeys given in literature 16
2.4 Permissible limits of trace metal concentration 18
3.1 List of honey samples collected from different sources 36
3.2 List of brand names and respective abbreviation of commercial
honey samples
36
3.3 Conditions for HPLC 39
3.4
Operational conditions employed in the determination of metals by
AAS
40
4.1 Fructose glucose ratio of Pakistani honey 71
4.2 Ca and Mg contents of studied Pakistani honeys 76
4.3 Iron, manganese and zinc contents of studied Pakistani honeys 80
4.4 Copper, lead and cobalt contents of studied Pakistani honeys 84
4.5 Cadmium, chromium and manganese contents of studied Pakistani
honeys
87
4.6 Isotopic ratio analysis of C12
/C13
in analyzed Pakistani honeys 92
4.7 Results of the principal component analysis on the physicochemical
properties
94
4.8 Result of analysis of variance for extraction of flavonoids by
different solvents
100
4.9 Mean zone of inhibition (mm) shown by honeys against gram
positive and negative bacteria
120
4.10 Mean MIC50 values of honeys V/V (%) gram positive and negative
bacteria
125
Glossary 165
Annexure-I Relationship of moisture contents with refractive index values of
honey
166
Annexure-II Color designation of honey samples: relationship of optical density
and Pfund scale
167
viii
LIST OF FIGURES
Figure No. Title Page NO.
2.1 Percent honey production of different countries 6
4.1 Electrical conductivity values of natural honeys from different
locations
49
4.2 Electrical conductivity values for adulterated, natural and
commercial honeys
51
4.3 Electrical conductivity values for honeys produced by Apis florea
(D) and Apis dorsata (G) collected from same locality
51
4.4 pH values for natural honeys collected from different locations 52
4.5 pH values of analyzed commercial Pakistani honeys 54
4.6 pH values of honeys produced by Apis dorsata (G) and Apis florea
(D) collected from same locality
54
4.7 Ash (%) of natural Pakistani honeys collected from different
location
55
4.8 Ash (%) of commercial Pakistani honeys 56
4.9 Ash (%) of honeys produced by Apis florea (D) and Apis dorsata
(G)
57
4.10 Moisture (%) of natural honeys collected from different localities
of Pakistan
58
4.11 Moisture (%) of commercial honey samples of Pakistan 59
4.12 Moisture (%) of honeys produced by Apis florea (D) and Apis
dorsata (G
60
4.13 Refractive index values of Pakistani honeys 61
4.14 Protein contents of natural honeys from different locations 62
4.15 Protein contents of some commercial Pakistani honeys 63
4.16 Protein contents of honeys produced by Apis florea (D) and Apis
dorsata (G)
63
4.17 Total free amino acid and total proline contents of honeys from
different locations
64
4.18 Total free amino acid and proline contents of honeys collected from
bee keepers
65
4.19
Total free amino acid and total proline level of some branded
commercial Pakistani honeys
65
4.20 Total free amino acid and total proline contents produced by Apis
florea (D) and Apis dorsata (G)
67
4.21 Sugar profile of natural honeys from different locations 67
4.22 Sugar profile of commercial honeys collected from bee keepers 68
4.23 Sugar profile of commercial honeys of local brands of Pakistan 69
4.24 Sugar profile of commercial honeys produced by Apis florea (D) 70
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Figure No. Title Page No.
and Apis dorsata (G)
4.25a HPLC chromatograms obtained for standards 72
4.25b HPLC chromatograms obtained for bee keeper’s honey samples 73
4.25c HPLC chromatograms obtained for some natural honey samples 74
4.26 Biplot of principal componenet analysis of natural, branded and
beekeeper’s honey samples
93
4.27 Dandogram of cluster analysis 95
4.28 Total phenolic contents of natural Pakistani honeys collected from
some selected locations
97
4.29 Total phenolic contents of commercial honeys of Pakistan 99
4.30 Total phenolic contents of natural honey produced by Apis florea
(D) and Apis dorsata (G)
99
4.31 Total flavonoids contents of natural honeys from different locations 101
4.32 Total flavonoids contents of honeys collected form beekeepers 101
4.33 Total flavonoids contents in honeys of different brands 102
4.34 Total flavonoids of honeys produced by Apis florea (D) and Apis
dorsata (G)
102
4.35 Vitamin C contents in natural Pakistani honey samples of different
location
103
4.36 Vitamin C contents in honey samples collected from some bee
keepers of Pakistan
104
4.37 Vitamin C contents in honey samples some local brands available
in markets of Pakistan
104
4.38
Vitamin C contents in honey samples produced by Apis florea (D)
and Apis dorsata (G)
105
4.39 β- Carotene contents of natural honeys 106
4.40 β- Carotene contents of commercial honey collected from bee
keepers
106
4.41 β- Carotene contents of commercial honey collected from local
markets
107
4.42 β- Carotene contents of honeys produced by Apis florea and Apis
dorsata
107
4.43 Lycopene contents of studied Pakistani honeys 108
4.44 Lycopene contents of Pakistani honeys Apis florea (D) and Apis
dorsata (G)
109
4.45 Ascorbic acid and qurecitin equivalent antioxidant profile of natural
honeys from different locations
110
4.46 Antioxidant profile of commercial honeys, adulterated honey and
mean of natural honeys
110
4.47 Antioxidant profile of Apis florea and Apis dorsata 111
4.48 Radical scavenging activity of honeys from different locations 113
4.49 Radical scavenging activity of standard, adulterated, mean of
natural and commercial honey samples
113
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Figure No. Title Page No.
4.50 RSA (%) of honey samples produced by Apis florea (D) and Apis
dorsata (G)
114
4.51 Ferric reducing antioxidant power of natural Pakistani honeys from
differernt localities
115
4.52 Ferric reducing antioxidant power of commercial Pakistani honeys 116
4.53 Ferric reducing antioxidant power of Apis florea and Apis dorsata 116
4.54 Inhibition (%) of peroxide by natural honeys from different
locations
117
4.55
Peroxide Inhibition (%) of standard, mean of natural and
commercial honey samples
118
4.56 Peroxide inhibition (%) of honey samples produced by Apis florea
and Apis dorsata
119
4.57a Pettry plates showing the zones of inhibition produced by some
honeys
123
4.57b Pasteurella multocida growth produced by some bee keeper’s
honeys
124
4.58 Pettry plates of potato disk assay of some honey samples 128
4.59 Inhibition (%) of tumors by different types of Pakistani honeys 129
4.60 Tumor formation in disk of positive (I) control, negative control
(II), sample with excellent tumor inhibition (III) and sample with
no tumor inhibition (IV)
130
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ABSTRACT
Nature has blessed us with large number of food stuffs honey is one of them. It is
composed of variety of substances mainly sugars, moisture and bioactive constituents. Its
composition depends on geographical and botanical origin. Physicochemical analysis is a tool for
quality evaluation, detection of adulteration and geographical discrimination of honey. Honey is
also reported to exhibit excellent bioactive composition which is responsible for therapeutic
behavior of this valued natural product. Therefore, present study was designed to evaluate
physicochemical properties, bioactive composition, antioxidant profile, antimicrobial behavior
and antitumor potential of honey. Honey was also used as bio indicator to detect the heavy metal
toxicity of some areas of Pakistan. Natural honey samples were collected from some selected
locations of Punjab, Pakistan. Beekeeper’s and branded honey samples were collected from local
markets of Pakistan. Quantitative, qualitative and medicinal properties of honey samples were
evaluated by using spectroscopic and chromatographic techniques. Nutritional values of all
Pakistani honey samples assessed by physicochemical analysis helped to conclude that all of our
honey samples obeyed international standards. EC values of beekeeper’s honey samples were
close to those found for natural Pakistani honeys. Natural and commercial Pakistani honeys
showed higher protein content as compared to available international data. Proline is the major
amino acid of honey. Natural Pakistani honey samples were excellent as compared to branded
and beekeeper’s honey samples. HPLC analysis of fructose and glucose ratio and GCMS
analysis of isotopic ratio analysis of C13
/C12
highlighted the adulteration in commercial and
branded honey samples. Trace metal concentration (Ca, Mg, Fe, Mn and Zn) of all analyzed
honey samples were also comparable with available data. In case of toxic metals many regions
like Faisalabad, Multan, Bahawalpur, Abdulhakeem and Dunyapur were found to contain toxic
metals more than permissible levels; lead and chromium being more prominent. Almost no metal
toxicity was found in commercial honeys. Total phenolic, total flavonoids, ascorbic acid, beta
carotenoids and lycopene were quantitatively found in natural honey samples in much higher
amount as compared commercial honey samples which contained in low amount. All studied
honey samples have shown excellent DPPH radical scavenging power, ferric reducing
antioxidant power and peroxide scavenging activities. Natural Pakistani honeys produced zones
of inhibition in the range of 10-28 mm for all tested bacterial strains whereas antifungal potential
observed against all analyzed strains was very poor. Many Pakistani honeys have excellent
antitumor potential as compared to many plant extracts. Some natural samples have antitumor
activity close to the standard and even two samples have shown more than standard. Many
commercial honey samples also have appreciable antitumor activities. Overall Pakistani honeys
were found to be promising natural products with excellent nutritional and medicinal properties.
1
CHAPTER 1 INTRODUCTION
Nature has blessed us with a large number of food materials; honey is one of them
and certainly the only sweetening material that can be consumed without any processing
(Vanhanen et al., 2011; Batista et al., 2012). Honey bees collect carbohydrate containing
excretions of plants and add several components including different enzymes that catalyze
biochemical changes and process the plant excretions to minimize simultaneously its
moisture contents (Guler et al., 2007; Rizelio et al., 2012; Consonni et al., 2013). Thus, some
constituents of honey composition are obtained from the plants, others are mixed by
honeybees, and yet some others produce after biochemical reactions during honey maturation
stages. Internationally, a lot of work has been done and reported on the chemical and
medicinal characterization of honeys (Campillo et al., 2012). Chemical constituents of honey
such as amino acids, sugars, aroma compounds, flavor (Pereira et al., 2008; Spano et al.,
2008; Kaskoniene et al., 2010; Kassim et al., 2012) phenolic compounds (Guerrini et al.,
2009; Jaganthan and Mandal, 2009; Zhou et al., 2102), flavonoids, (Kenjeric et al., 2007; Al
et al., 2009; Guerrini et al., 2009; Tenore et al., 2012; Oroian, 2013) ascorbic acid, aliphatic
compounds, organic acids and analysis of norisoprenoids and terpenoids have also been
proposed to verify the authenticity of honey (Tornuk et al., 2013).
Nutrient Data Laboratory of United States (NDLUS) reported a detailed nutritional
composition of a variety of floral and other types of US honeys which provided useful
information to chemists and food technologists. Several studies evidenced European honeys
to have a rich phenolic profile, consisting of benzoic, cinnamic acids and flavonoid
aglycones (Martos et al., 2000; Yaoa et al., 2003).
International Honey Commission of Food and Agriculture Organization of the
United Nations reported that along with organic nutrients honey also contains some mineral
components like sodium, potassium, calcium, zinc, copper, magnesium, manganese,
selenium, iron, chromium and phosphorous (Kujawski and Namiesnik, 2008; Khuder et al.,
2010). The George Mateljan Foundation includes honey in world‟s healthiest foods.
2
Adulteration of cane sugar, commercial invert sugar, corn syrup and other sugars is
very common in honey (Guler et al., 2007; Mahindru, 2007; Schellenberg et al., 2010; Boffo
et al., 2012; Rizelio et al., 2012). However, studies have shown that some chemical
characterization could be used to distinguish between pure and adulterated honeys.
Carbohydrates, amino acids, flavonoids, and mineral contents have been valuable parameter
for identifying pure and impure honey samples (Guler et al., 2007; Truchado et al., 2008;
Truchado et al., 2010). Analysis and comparison of chemical and physical properties are
useful tools to highlight differences among natural, beekeeper‟s and branded commercial
honeys which might be helpful for quality evaluation of various types of honeys by their
comparison with international standards of honey like CODEX standards adopted in 1981
and then revised in 1987 and 2001, United States standards for honey grading adopted in
1985. As quality of honeys from different locations varies due to botanical variations,
therefore detailed chemical characterization is helpful to detect quality differences among
honeys produced in different origins.
Nutritional composition of honey depends on environmental biotic (plants) and abiotic
(soil, water and air) factors. Honey bees roar around its environment for nectar collection and
are exposed to a variety of pollutants present in soil and water. Hence honey bees and their
products can be used as bioindicators (Bogdanov et al., 2003; Khuder et al., 2010; Chauzat et
al., 2012; Ru et al., 2013) to detect environmental pollution in different origins native to
Pakistan, qualitatively and quantitatively.
Honey is not only important due to its unique composition but also due to its different
therapeutic potential (Parmar et al., 2013). Therefore, it is largely produced and characterized
by many countries of the world.
Antimicrobial properties of honey are attributed to low moisture levels (14-18%) of
honey as it absorbs the moisture from microbes and kills them due to dehydration. Chemical
and medicinal characterization of honeys from different sources has been published by many
researchers around the globe (Rodrıguez et al., 2004; Ahmed et al., 2007; Guerrini et al.,
2009; Aliferis et al., 2010; Ayaad et al., 2012).
3
Antioxidants play a vital role against the deteriorating effects of free radicals in the
organisms. Deficiency of antioxidants in living organisms leads to oxidative stress. Chemical
and medicinal research based on antioxidants from fruits, vegetables, spices, herbal teas and
beans etc. has gained tremendous importance recently as scientists are interested to substitute
the synthetic antioxidants by their natural counterparts. Botanical sources like berries,
cherries, black grapes, kiwi, pomegranates, mangoes, citrus fruits, prunes, plums, apples,
pears, parsley, yellow onion, oats, wheat, rice etc. are known to have one or more
antioxidants like flavanols, hydroxyl benzoic acids, hydroxycinammic acids, anthocyanins,
p-coumaric acid, ferulic acids, caffeic and catechins (Beecher, 2003; Manach et al., 2004;
Dimitrios, 2006; Liu et al., 2013). Natural antioxidants are also preferred over synthetic ones
as food preservatives due to their toxic effects. Natural antioxidants found in honey are
effective against inflammation, cancer, tumor, coronory diseases, burns, aging, wound
healing, gastrointestinal and heart diseases (Rodrıguez et al., 2004; Ahmed et al., 2007;
Guerrini et al., 2009; Aliferis et al., 2010; Ayaad et al., 2012; Keckes et al., 2013). Honey
has also been proven to be an excellent natural antimicrobial, antiparasitic, antiviral, and
antimutagenic agent (Bogdanov et al., 2008). Antioxidant potential of honeys from different
origins varies due to botanical and geographical variations (Martos et al., 2000; Yaoa et al.,
2003) as discussed earlier. On the other hand, comprehensive data about antioxidant potential
of Pakistani honeys are also not available. So, it becomes important to characterize
antioxidant properties of honey samples from different origins of Pakistan.
Previous studies suggested that various types of honeys from different geographical
origins have shown antioxidant profile depending on the quality and quantity of different
bioactive compounds. It was reported that many phytochemicals present in honey are
responsible for free radical scavenging activity (Aljadi and Kamaruddin, 2004). Among
those phytochemical flavonoids present in honey are also responsible for antioxidant
properties of honey (Ayaad et al., 2012). A study reported by Al-Mamary et al. (2002) has
suggested that different types of honeys from different origins of world have antioxidant
profile depending on the quantity of phenolic groups. Vitamin C (ascorbic acid) proved an
excellent antioxidant and even use as standard for antioxidant studies and catalase are also
present in honey (Vidal et al., 2009).
4
Therefore, antioxidant and antimicrobial properties of honey are due to presence of a
variety of enzymes and phytochemicals like flavonoids, phenolics, ascorbic acid, α-
tocopherol, gulucose oxidase and catalase etc. (Al-Mamary et al., 2002; Vidal et al., 2009;
Manyi-Loh, 2012). Two of the major antibacterial compounds identified in New Zealand
honeys were methyl 3, 4, 5-trimethoxy benzoate and methyl 4- hydroxy-3, 5-dimethoxy
benzoate. Other flavonoids of antibacterial activity were also detected in honeys from
different localities of Spain and Argentina. Of these flavonoids, pinocembrin was detected
whereas kaempferol and quercetrin, as well as pinocembrin and naringenin were detected in
sunflower honey. The presence of chrysin and galangin in several Swiss honeys has been
reported. The best techniques for analysis of flavonoid contents of honey are HPLC, GC,
LCMS and GCMS (Kenjeric et al., 2007; Guerrini et al., 2009; Truchado et al., 2010). In
addition, many natural antibacterial compounds have been identified from different types of
honey floral honeys.
The antimicrobial and antibiotic studies of honeys have also been subjected to
extensive analysis. Agar-well diffusion, disc diffusion and spectrophotometric methods are
the most suitable for determination of antibacterial and antibiotic activities of honey (Gomes
et al., 2010; Gulfraz et al., 2010; Manyi-Loh et al., 2010; Atrott et al., 2012; Moussa et al.,
2012a). Limited work reported so far, advocate considerable anticancer potential of honey
both in vivo and in vitro (Fauzi et al., 2011; Shati et al., 2010). Analysis of Greek honeys
concluded that honeys with high phenolic contents showed estrogenic activities while thyme
honey rich diets are effective against prostate, endometrial and breast cancers (Tsiapara et al.,
2009). Moon et al. (2006) reviewed anticancer mechanisms of flavonoids and concluded that
flavonoids are effective against cytochrome P450 (CYP) enzymes involved in the activation
of procarcinogens and phase II enzymes, largely responsible for the detoxification of
carcinogens. Although honey had been reported as an excellent bioactive natural product but
unfortunately was not characterized for its antitumor activities in detail, therefore, it is
imperative to analyze the antitumor activities of honey (Jaganathan and Mandal, 2009).
Flavonoid, phenolic and amino acid profile of honeys has also been useful for
determination of botanical origin of honey as quality of honey is directly related to its
geographical and botanical origin. Pollen identification for discrimination of botanical origin
5
is not an authentic parameter because beekeepers artificially add pollens in beefed and
adulterated honeys. Earlier studies showed a correlation between botanical origin and
flavonoid profiles of honeys (Anklam, 1998).
Analytical investigations of the honeys from many countries have been carried out
and their compositional, nutritional and pharmaceutical data base has been established
indicating variation in these profiles depending upon their geographic and floral origins.
Such studies are seldom conducted in developing countries like Pakistan. Although limited
research has been carried out on some selective honey samples from Pakistan in the past but
there is no complete chemical and medicinal data available for various types of natural and
commercial Pakistani honeys. It has been reported by Pakistan Science Foundation that 80%
of available Pakistani honeys are adulterated so it is, therefore, necessary to discriminate the
impure stuff from pure Pakistani honeys. Consequently, the present study was focused on the
evaluation of the chemical and medicinal worth of Pakistani honeys with respect to
international standards. We also compared nutritional, antitumor, antimicrobial and
phytochemical profile of natural and commercial Pakistani honey. This will help in
establishing comprehensive baseline database for Pakistani honeys that will serve as a mile
stone for researchers in the related fields in future.
Aims and Objectives:
1. Evaluation of chemical, nutritional and medicinal parameters of Pakistani honeys.
2. Comparison of scientific protocols to check adulteration in the honeys.
3. Evaluation of mineral profile and heavy metals toxicity in honeys.
4. Appraisal of antimicrobial, antioxidant and anticancer activities of the collected honey
samples.
6
CHAPTER 2 REVIEW OF LITERATURE
The current study was planned to investigate the physicochemical and medicinal
properties natural as well as commercially available Pakistani honeys. Honey production in
the world is increasing day by day. China is the largest producer of honey and produced
398,000 metric tons annually which is 31.17% of total world production. Turkey is the
second largest producer with 81,115 metric tons of annual honey production. Amount of
honey produced by European Union was 22% of world honey. New Zealand is leader in
production of monofloral honeys. Percent honey production by different countries
(Australian Government, 2008; Isla et al., 2011) is given in the Fig. 2.1.
Fig. 2.1: Percent honey production of different countries
In this scenerio, honey production in Pakistan has also been increasing rapidly due to
awareness regarding economic and health benefits of beekeeping technologies. Excellent
ecological conditions of Pakistan are valuable blessing of Allah Almighty for abundant flora
which in turn is helpful for beekeeper‟s to produce excellent grade honey. Pakistan is
producing 7,500 metric tons of honey each year only due to beehives of Apis mellifera and
still has multifold capacity (http://www.parc.gov.pk/honey.html). Pakistani honeys of
0
1
2
3
4
5
6
% H
on
ey
Pro
du
ctio
n
Country
7
excellent quality are known for their health benefits, especially Ziziphus honeys are
considered as the most valuable honeys of the world (Gulfraz et al., 2010).
According to Belitz et al. (2004) honey production by bees starts with collection of
nectar from flowers and other sweet saps of plants. Honey bees add some substances from its
salivary glands and from walls of honey sacs (bees pouch) and store these plant juices in their
honey sacs for some time. Finally, bees reach to their bee hives and store this nectar in honey
combs where ripening processes of honey starts with evaporation of water from nectar to
make it viscous followed by hydrolysis of sucrose in presence of bee‟s enzymes and organic
acids which lead to production of invert sugars. Further enzymatic isomerization of glucose
to fructose takes place. Honey bees also produce heat in the hive by movement of their wings
to evaporate most of the water until the moisture content reduces to 15- 23%. Honey storage
cells are covered with wax lids. Ripening process continues with glucose to fructose
conversion and production of some new sugars by floral invertases. After ripening honey is
removed from combs either by extraction, pressing, straining or pulping. Codex Alimentarius
Standards assigned the following definition to honey “Honey is the natural sweet substance
produced by honey bees from the nectar of plants or from secretions of living parts of plants
or excretions of plant sucking insects on the living parts of plants, which the bees collect,
transform by combining with specific substances of their own, deposit, dehydrate, store and
leave in the honey comb to ripe and mature” (Codex Alimentarius Commission, 2001). The
highest quality honey is obtained and enjoyed in pure form is also called domestic honey.
Honey can be classified into two broad groups on the basis of it sources: floral honeys and
honey dew honeys. Floral honeys are produced from nectar of flowers like heather, linden,
acacia, meadow white clover, sweet clover, alsike, buckwheat, rape, citrus, eucalyptus and
many more. Floral honeys are available in wide range of colors like white, greenish yellow,
brownish, light to dark, amber, dark red or reddish amber. On the other hand, honey dew
honeys are produced by collection of not only nectar but also plant exudates and sugary
excretions of homopterous insects which these insects excrete on the surface of plants during
their visits.
8
Table 2.1: Basic composition of honey for authenticity of honey
Composition Canadaa Caribbean
Communityb
FAO/WHOc
dThe Honey
Regulations
Apparent reducing sugar (%) > 60-65 60 > 60 60
Moisture (%) 20 >20 > 23 20
Apparent sucrose (%) 5-10 >5 > 10-15 5
Water- insoluble solids (%) 0.1-0.5 0.1 − 0.3 0.1-0.5 0.1
Ash (%) 0.6-1
Acid milliequivalents/ 1000 g 40 50 >50 50
Colour Pale to dark
amber
Electrical
conductivity(mS/cm)
>0.8 0.8 0.8
Hydroxymethylfurfuraldehyde >80 80 >40
a Honey Regulations (Canada), 2013, bCaribbean Community,2011,c Codex Alimentarius
standard by FAO/WHO, 2011 dThe Honey (England) Regulations, 2013
These honeys are less viscous and are not easily solidified, less sweet and darker in
color than floral honeys (Saxena et al., 2010). Honey is mainly composed of sugars and
moisture. Composition of honey reported by different standards of honey is given in the
Table 2.1(Codex Alimentarius Commission, 2001; USDA National Nutrient Database for
Standard Reference, 2011; The Honey (England) Regulations, 2013). Honey cannot be
consumed as complete diet but it can be added as dietary supplement (Silva et al., 2009).
USDA National Nutrient Database reported that honey (100 g) is composed of
Carbohydrates (82.4 g), Sugars (82.12 g), Dietary fiber (0.2 g), Fat (0 g), Protein (0.3 g),
Water (17.10 g), Riboflavin (0.038 mg), Niacin (vitamin B3) (0.121mg), Folate (2 μg),
Pantothenic acid (B5 0.068 mg), Vitamin B6 (0.024 mg), Vitamin C (0.5 mg), Calcium (6
mg), Iron (0.42 mg), Magnesium (2 mg), Phosphorus (4 mg), Potassium (52 mg), Sodium (4
mg) and Zinc (0.22 mg) (USDA National Nutrient Database, 2011). All these components
make a little contribution in overall composition of honey and daily recommended intake of
these components is not enough only with the consumption of honey (Bogdanov, 2008).
9
2.1 Species of honey bee
There are different species of bees which can produce honey and honey obtained
from either is valuable from therapeutic and nutritional purposes in all cultures. Apis dorsata
(rock bee), Apis mellifera (European or Italian bee), Apis ceranaindica (Indian hive bee),
Apis florea (little bee) and Melipona irridipennis (Dammer bee or stingless bee) are five
major species of honey bees active in honey production. Three honey bee species Apis
dorsata, Apis florea and Apis cerana; the species indigenous to Pakistan are found in
different ecological regions of Pakistan. Apis mellifera (imported specie) is now extensively
used by beekeepers in Pakistan (http://www.parc.gov.pk/honey.html). Pakistani population
considered Apis floreal (little bee) honey beneficial therapeuticaly as compared to the honey
produced by Apis dorsata without any experimental evidence. Therefore, both types of
honeys should be analyzed chemicaly and medicinaly.
2.2 Physicochemical characterization
Biotic (bee species and botanical sources) and abiotic (geographical and climatic
conditions) are the major factors of environment which collectively determined the
composition and functional quality of honey (Paradkar and Irudayaraj, 2001; Downey et al.,
2005; Malika et al., 2005; Ouchemoukh et al., 2007; Ajlouni and Sujirapinyokul, 2010;
Gomes et al., 2010; Kaskonienene et al., 2010). Physicochemical properties and bioactive
potential of honey is necessary to characterize the quality of honey. Several studies on
honeys from many locations demonstrated that physicochemical parameters an be used for
characterization, classification and authentication of the honeys (Rodriguez et al., 2004;
Soria et al., 2004; Corbella and Cozzolino, 2006; Gomes et al., 2010; Kristina et al., 2012;
Escriche et al., 2012). Identification of botanical origin of honeys by melissopalynological
(pollen counting) is a laborious and time consuming technique which required special
knowledge and expertise for interpretation of results. On the other hand,
melissopalynological analysis is not very accurate method due to artificial addition of pollens
by beekeeper to misguide consumer (Bianchi et al., 2005; Kaskoniene and Venskutonis,
2010; Tiwari et al., 2013; Ulloa et al., 2013). Irish honeys were classified into geographic
and botanical origins on the basis of physicochemical properties of honeys (Downey et al.,
10
2005). Andalusian honeys of Spain were characterized on the basis of physicochemical
parameters. Application of statistical tools helps to identify six parameters which can use
with high confidence for classification of Spanish honeys. These six parameters were
moisture%, acidity, total sugars, total solids, invertase and electrical conductivity (Serrano et
al., 2004). A study conducted on Argentinian honeys has employed twenty two
physicochemical parameters to discriminate the honeys on geographical basis (Baroni et al.,
2009). These studies concluded that six physicochemical parameters including pH, glucose,
free amino acid, free acidity, zinc and calcium can be used very confidently to assign
geographical location and authentication to their honeys. Another study helped to
discriminate provenance of honeys on the basis of physicochemical characteristics like
acidity, electrical conductivity, sugar profile, amino acid and proteins profiles of honey.
Honeys from Uruguay (a region of South America) were also classified into floral origins on
the basis of some physicochemical parameters (Baroni et al., 2004; Cometto et al., 2006;
Cimpoiu et al., 2013; Yuceln et al., 2013). Corbella and Cozzolino (2006) suggested that
more physicochemical parameters should be analyzed for geographical classification of
honeys Uruguayan honeys. Physicochemical values determined for the honey from different
origins of Venezuela were significantly different (P≤0.05) (Rodriguez et al., 2004). Kamal et
al. (2002) analyzed some physicochemical parameters of honeys collected from Islamabad
(Pakistan) for identification of floral origin. Physical parameters like color, ash (%),
electrical conductivity, refractive index, viscosity, turbidity, optical activity and pH etc are
excellent tools to discriminate both botanical as well as to geographical origin of honeys
(Soria et al., 2004; Corbella and Cozolino, 2006; Guerrini et al., 2009). Downey et al. (2005)
used six physicochemical properties and mineral composition for characterization of honey
samples produced on island of Ireland. Although all analyzed honeys were characterized as
floral origin but that information was insufficient for authentic classification of their honey
samples.
Therefore, it can be hypothesized that all parameters are not equally important to
assign geographical location and botanical origin to honey. However a detailed
physicochemical analysis and comparison of the values with international literature can
provide basic compositional evidence which help to discriminate the localities within the
same origin, to highlight adulteration and to check the accordance with data published by
11
different countries. Brief introduction, chemistry and values of most important
physicochemical properties of honeys are discussed below.
2.2.1 Ash and electrical conductivity (EC)
Ash content represents the inorganic residues obtained after carbonization and a
criterion of specific botanical origin of honey (Downey et al., 2005). Honeydew honeys and
blossom honeys were previously classified on the basis of percent ash of honey. Blossom
honeys contain ≤ 0.6% ash while honeydew honeys contain ≤ 1.2% ash. EC value is directly
proportional to ash% of the honey. On the other hand, ash contents also depend on the
mineral contents of honey (Ouchemoukh et al., 2007). Electrical conductivity values of
honey are found to have strong correlation (r2=0.973) with mineral content of honey
(Vanhanen et al., 2011). Electrical conductivity value of honey depends not only on ash but
also some other components like proteins, polyol content and acids which in turn depend on
the botanical origin of honey. Codex Alimentations standards of honey recommended to use
EC values instead of ash contents as a parameter to determine the origin of honeys
(Kaskoniene et al., 2010). According to European Economic Community EC is among the
parameters that can be used to discriminate honeydew honeys and blossom honeys. Blossom
honeys exhibited less than 0.80 mS/cm while honeydew honeys exhibited more than 0.80
mS/cm EC (European Economic Community, 2002; Manzanares et al., 2011).
2.2.2 Color of honey
According to Brudzynski and Miotto (2011), many compounds like phenolics,
flavonoids, isoprenes, terpenes, carotenoids and lycopenes make the honey colors in range of
yellow to brown. All these compounds are also excellent antioxidant agents. This is why
darker honeys exhibited higher antioxidant potential as compared to lighter honeys. On the
other hand, maillard reaction (Non enzymatic browning) is also thought to be responsible for
color of honey in addition to above mentioned compounds. These compounds also exhibited
antioxidant potential. Correlation of color of twenty honey samples (collected commercial
markets and apiaries of Canada) with oxygen radical scavenging capacity, total phenolic
contents and Maillard reaction products was developed. Honey color was determined by
12
Table 2.2: Relationship of honey color grade and Pfund scale
Color Designations Color Range Pfund (mm) Optical Density
Water White Water White or lighter darker than Amber 0.0945
Extra White Darker than Water White,
but not darker than Extra
White.
Over 8 to 17 0.189
White darker than Extra White,
but not darker than White
Over 17 to 34 0.378
Extra Light Amber darker than White, but not
darker than Extra light
Amber
34 to 50. 0.595
Light Amber darker than Extra Light
Amber but not darker than
light Amber
50 to and 85 1.389
Amber darker than light Amber,
but not darker than Amber
85 to 114 3.008
Dark Amber darker than Amber Over 114 …….
taking absorbance at 560 nm with the help of spectrophotometer and color grades were
assigned from table of honey standards of USDA. Excellent correlation in the range of 0.86
to 1.000 was found between honey color, total phenolic contents, Maillard reaction products
and oxygen radical scavenging power. Honey is graded on the basis of its color and optical
density by USDA standards. Honey color is graded on a scale called the Pfund scale. USDA
standards for honeys have also developed a relationship of honey color on the basis of optical
density measurement at λmax 560 nm. This relationship was shown in Table 2.2 and gives
information of grades of color as darkness/lightness, mm Pfund, and color ranges on the basis
of optical density measurements. Literature survery concluded that many studies on honey
color rely on the values given in that USDA table of color measurement (National Honey
Board, 2005; Al et al., 2009; Isla et al., 2011).
13
2.2.3 pH
Honey is acidic in nature due to presence of inorganic ions like phosphates and
chlorides and due to some organic compounds like, organic acids; acetic acid, butyric acid,
gluconic acid, citric acid, maleic acid, lactic acid, succinic acid, oxalic acid, glycolic acid,
alpha-ketoglutaric acid, pyruvic acid, tartaric acid and amino acids. Honeydew honeys are
more acidic as compared to blossom honeys. Formic acid once considerd as “the acid of
honey” because it was the 1st organic acid found to be present in the honey, but in minor
quantity (Mahindru, 2007). Honey pH has also been found to have high correlation
(r2=0.776) with mineral content of honey (Vanhanen et al., 2011).
2.2.4 Moisture contents
Honey moisture percentage varies with geographical, seasonal, floral and even with
beekeeper‟s handling processes (Ouchemoukh et al., 2007). Maximum permitted value of
honey moisture is 21% by National Honey Board of European Commission and 20% by
Codex Alimentarius Standards of honey. In case of heather honeys, moisture level may rise
up to 23%. Low moisture level makes it stable against microbes (Malika et al., 2005). On the
other hand, no biochemical reactions start at low moisture level which leads to the stable
composition for long periods of time (European Economic Community., 2002; Food
Standard Autralia and New Zealand, 2006). High moisture level may lead to spoilage, flavor
loss or fermentation reactions in the honey (Downey et al., 2005). Moisture percentage too
determines the quality of honey. Moisture level of honey can be determined by refractive
index while refractive index of honey is different as compared to the sucrose solution.
Therefore, a special chart recommended by AOAC is used for determination of moisture
contents with the help of refractive index measurements (National Honey Board, 2005).
2.2.5 Proteins and free amino acids
Botanical sources and honeybee glands are responsible for honey protein and amino
acid (Hermosin et al., 2003). Variety of enzymes like invertase, α-amylase, glucose oxidase,
phosphatase and catalase contributed by honeybee saliva, nectar and pollens are responsible
for total protein of honey (Kucuk et al., 2007; Saxena et al., 2010). About 1% of honey
weight is composed of proteins and free amino acid. There are about twenty seven amino
14
acids present in honey. It is even more authentic to use free amino acid as compared to
protein as botanical and origin marker. Some amino acids are thought to be responsible for
aroma and flavor of honey (Hermosin et al., 2003). Estimation of amino acids is important
due to nutritional labeling (Pereira et al., 2008). Quantitatively the most abundant amino acid
of honey is proline (Meda et al., 2005; Paramas et al., 2006). Ouchemoukh et al. (2007)
found that correlation between proline quantity and radical scavenging activity is higher as
compared to the correlation between radical scavenging activity and phenolic contents of
honey. Although good correlation was found between color of honey and ascorbic acid
equivalent antioxidant contents, ferric reducing antioxidant power and DPPH radical
scavenging activity, however, excellent correlation of 0.96 and 0.94 was found between
proline load and DPPH scavenging activity and proline content and ascorbic acid equivalent
antioxidant contents, respectively. Proline was reported as major factor responsible for
overall antioxidant potential of analyzed Indian honeys (Saxena et al., 2010). Proline has also
been found an as indicator not only for ripeness but also for purity of honey (Bogdanov et al.,
1999: Hermosin et al., 2003; Guler et al., 2007; Baroni et al., 2009). Proline contents of
honey vary with botanical origin and specie of honey bee. A study conducted to classify
seventy seven honey samples from the Spain revealed that proline contents of honeydew
honeys and blossom honeys differed significantly. Amount of proline contents honeydew
honeys and blossom honeys were 1069±284 and 602±140 mg/kg, respectively (Manzanares
et al., 2011). Proline ontents of floral honeys determined by Gulfraz et al. (2010) were
1.25±0.06, 1.82±0.03, 2.1±0.04 and 1.96 mg/kg for acacia, brassica, citrus and ziziphus
honeys, respectively. Ilex, oak, chestnut-tree and heather honey contained 49.5, 37.6, 29.6
and 49.6% proline out of total free amino acids.
2.2.6 Hydroxymethylfurfuraldehyde (HMF)
Heat processing of foods rich in monoschrides is liable to production of
hydroxylMethylfurfuraldehyde (HMF). HMF production is proportional to heat treatment
and obeys the pseudo first order reaction. HMF in small quantity is always present in honey
due to acid catalyzed and enzymatic dehydration of monosacchardes. Large amount of HMF
in the honey is an indication of mishandling of beekeepers to make adulteration by invert
syrup (Wong et al., 2012; Kowalsk, 2013). Pentoses and hexoses on heating are dehydrated
15
to aldehydes e.g. Hexoses are dehydrated to 5-hydroxymethyl furfural (HMF). Reaction is
given below.
O
OH OH
OH
OCH2OH-CHOH-CHOH-CHOH-CHOH-CHO
Hexose Sugar HMF
HMF is the criteria of purity, freshness and quality of honey by Codex Alimentarius
and EU standards of honey (Downey et al., 2005). Maximum permissible value of HMF in
honey by the Codex Alimentarius is 80 mg/ kg while by the EU standards it is 40 mg/ kg
(Finola et al., 2007; Turhan et al., 2008; Manzanares et al., 2011). HMF can be identified by
addition of m-hydroxyl benzene in presence of hydrochloric acid. Reaction is given below
(Mahindru, 2007).
O
O
OH OH
OH
+
OH
OHO
OH OH
OH
OH
OH
+
HMF m-hydroxyl benzene Cherry red Condensation product
It will form a cherry red condensation product. Therefore, this test can be used to
detect the HMF as a quality tool this valued natural product.
2.2.7 Sugar profile of honey
On the basis of dry mass, 95% of honey is composed of sugars with
monosaccharaides being the principal component. Invert sugar fructose and glucose
constituted major composition of honey sugar (Boffo et al., 2012). About 3% of requied
energy can be covered with consumption of 20 g of honey (Bogdanov, 2008). Fructose is
major monosaccharide of honey except some varieties of monofloral honeys like Brassica
16
napus, Taraxanum officinale and Trichostema lanceolatumi which are richer in glucose.
Glucose is less water soluble as compared to fructose therefore high glucose level lead to
Table 2.3: Sugar profile of different types of honeys given in the literature
Type of
Honey
Fructose
(g/100 g)
Glucose
(g/100 g)
Maltose
(g/100 g)
Reference
Willow 32.92±2.20 to
38.88± 2.40
35.270± 0.90 to
42.29 ± 0.80
11.4 ± 0.1 to
18.5 ± 0.6
Kaskoniene et al.,
2010
winter rape 34.42 ± 1.4 to
35.47 ± 3.70
40.36 ± 3.0 to
42.63 ± 0.6
0.59 ± 0.1 to
1.57 ± 0.60
Kaskoniene et al.,
2010
Spring rape 35.14 ± 2.6 to
37.84 ± 0.70
40.36 ± 3.0 to
42.63 ± 0.6
0.50 ± 0.20 to
2.64 ± 0.2
Kaskoniene et al.,
2010
White clover 35.85 ± 1.8 to
40.00 ± 1.1
34.60 ± 1.70 to
41.21 ± 0.50
0.58 ± 0.30 to
1.24 ± 0.40
Kaskoniene et al.,
2010
blossom
honeys
39.54±1.20 33.29±1.96 5.52±0.88 Soria et al., 2004
Honeydew 39.64±1.71 31.19±2.33 5.52±0.89 Soria et al., 2004
granulation and hardness during long storage and cold weather (Rodriguiz et al., 2004).
Sugar profile of honey depends on origin of honey. Sugar profile of different types of types
of honeys is given in the table.
Fructose glucose concentrations and their ratios are helpful to check the authenticity of honey
especially in case of bee feeding of with glucose and sucrose syrups by the bee keepers
(Ouchemoukh et al., 2007; Kaskoniene et al., 2010; Rizelio et al., 2012). Average F/G ratio
is 1.2/1 (Rodriguez et al., 2004). Codex Alimentarius standards of honey allowed minimum
1.14/1(F/G) (Codex Alimentarius Commission, 2001).
2.2.8 Isotopic ratio analysis for detection of adulteration
Addition of high fructose corn syrups (HFCS) is a sophisticated technique which has
no effect on natural fructose glucose ratio of natural honeys (Fairchild et al., 2003; Puscas et
17
al., 2013). Stable isotopic ratio analysis of elements has gained importance not only in
detection of adulteration but also for determination of the geographical origin of food
materials (Schlicht et al., 2006; Camin et al., 2007; Rossmann and Schlicht, 2007). Most of
the sweeteners are originated from corn and cane sugars. Corn and cane plants are classified
as monocotyledonous plants. On the other hand, almost all flowering plants are dicotyledons.
Photosynthetic reactions in C3 (flowering) plants takes place by Calvin–Benson cycle which
leads to C13
/C12
ratio in the range of -32 to-21% while in C4 (corn and cane) plants Hatch-
Slack cycle of photosynthesis leads to production of C13
/C12
ratio in the range of -19 to -12%.
Adulteration of corn and cane sugars in natural honeys disturbs original C13
/C12
of natural
sugars originated from flowering plant (Padova n et al., 2003; Kropf et al., 2010;
Schellenberg et al., 2010). Therefore, isotopic ratio analysis can be used with confidence for
detection of artificial sweetners in the honey.
2.2.9 Metal contents
Presence of high mineral levels in Pakistani plants guide honey production with high
mineral contents and make them very important food nutritionally especially for children.
High iron, zinc, manganese, calcium and potassium may significantly contribute to the daily
intake of metals (Gulfraz et al., 2010). Approximately 0.1 to 0.2% of minerals are present in
floral honeys while 1% or higher mineral find in mellate or honeydew honeys (Hernandez et
al., 2005; Bilandzic et al., 2011). Potassium has been reported as the most abundant and
sodium as second most abundant metal in honey followed by Ca and Mg while Cu, Rb, Mn,
Fe and Zn are in intermediate amount. Potassium contributes about 45-85% of total metal
profile of honey although its concentration varies with region. (Silva et al., 2009; Batista et
al., 2012; Alves et al., 2013). A study conducted by Silici et al. (2008) deduced that trace
elements play both positive as well as negative role in human health therefore; honey can
serve as an important source of trace elements. Thus analysis of mineral profile of honey
becomes imperative to assess its nutrition potential as mineral supplement (Vanhanen et al.,
2011). In addition, it may also be helpful to assign geographical and botanical origin as well
as to detect the level of possible environmental pollution of area where bees hive for nectar
(Badiou et al., 2008; dos Santos et al., 2008; Pisani et al., 2008; Bilandzic et al., 2011;
Badiou-Beneteau et al., 2012; Lambert et al., 2012a; Lambert et al., 2012b; Barganka et al.,
18
2013; Ru et al., 2013). Overall twenty seven mineral elements in honey have been reported
so far from nine countries but each honey contributed a few of them individually (Baroni et
al., 2009; Gulfraz et al., 2010; Fermo et al., 2013). Table 2.3 showed the values for
permissible limits of metals recommended by Czech by law in certified reference material
(Celechovska and Vorlova, 2001; Fowler et al., 2007; Silici et al., 2008). Analysis of these
toxic elements in nutritional products is a valuable tool which is not only useful to detect the
metal load in natural food products but also applicable for evaluation of concentration and
type of toxic metals in surrounding environment (Pisani et al., 2008; Rashed et al., 2009;
Bilandzic et al., 2011). A study on Croatian honeys was conducted to evaluate the heavy
Table 2.4: Permissible limits of trace metals
metal toxicity of specific metals from some regions of Croatia. Five metals Cu, Pb, Hg, As
and Cd were quantified from five locations of Croatia. Overall lead (108 µg/kg) and copper
(131 µg/kg) were found to be the most abundant toxic metals in Central region, As (23.8
µg/kg) in honey samples of south region, Hg (2.63 µg/kg) in the northeast region and Cd
(2.11 µg/kg) in the southwest region of Croatia. That study deduced that lead contents were
at the highest level as compared to the honeys from other European countries. Furthermore,
analyzed metal constituents from one area were significantly different from other areas. It
was suggested to build beehives far away from railway tracks and highways (Bilandzic et al.,
2011). In another study, arsenic a pollutant was found in much higher concentrations in
honeys from an area of Portuguese due to burning activities in surrounding areas (Silva et al.,
2009). To the best of our knowledge there was reported no work so far on Pakistani honey
with regard to heavy metal pollution especially in the indutrial zones.
Metal Certified Level (µg/g) Metal Certified Level (µg/g)
Cu 5.64 Se 0.05
Cd 0.013 Zn 12.50
Pb 0.47 Mn 54.0
Co 0.09 Fe 83.0
Cr 0.30 K 16.10
Ni 0.91 Ca 15.26
Al 2.86 Mg 2.71
19
2.3 Bioactive composition of honey
Honey is a source of bioactive compounds with more than 200 constituents/
phytochemical like vitamins, proteins, organic acids, phenolic, minerals, enzymes,
flavonoids, volatile compound, alkaloids, free amino acids, carotenoids, Maillard reaction
products and many more (Gheldof et al., 2003; Escuredo et al., 2013; Keckes et al., 2013;
Tornuk et al., 2013). Botanical origin and bee species are the major factors responsible for
determination of bioactive composition of honey. Floral source is dominant parameter for
quantitative and qualitative evaluation of bioactive constituents of this valued natural
product. Therefore, analysis of bioactive composition is an excellent tool to characterize the
honey for its medicinal importance from different botanical and geographical origins (Malika
et al., 2005; Al et al., 2009; Ramanauskiene et al., 2012; Zhou et al., 2012). More than 50%
of total land area of Pakistan is under cultivation. Due to its geographical conditions it has
also rich source of medicinal plants, which are widespread over a range (Ali, 2008; Shinwari
and Shinwari, 2010). Keeping in view these conditions, it is expected to have high quality
honey with excellent bioactive composition however no study on exploration of bioactive
composition for Pakistani honeys has so far been reported to our knowledge. Present study
paid a special attention to the analysis of bioactive compounds like total phenolics, total
flavonoids, total free amino acid, total proline, ascorbic acid, and carotene and lycopene
contents for Pakistani honeys. The antioxidant activities have also been determined and all
being reported here.
2.3.1 Phenolics
Polyphenols and flavonoids are the plants secondary metabolites. Phenolic acids and
flavonoids (cinnamic acid and benzoic derivatives) are the most important classes of plant
polyphenol. More than 5000 phenolic compounds have been reported (Pyrzynska and
Biesaga, 2009). Honey mainly contained more than 150 phenolic compounds including
flavonoids, flavonols, catechins, stilbenes, phenolic acids, oxidized polyphenolics, lignans,
cinnamic, tannin acid derivatives etc. There are many studies reporting quantitative and
qualitative analysis of honey phenolics (Baltrusaityte et al., 2007; Buratti et al., 2007;
Devarajan and Venugopa, 2012; Escuredo et al., 2012; Biesaga and Pyrzynska, 2013). Total
phenolic compounds can be determined by Folin-Danis method, colorimetric method of iron
20
complex, titration with permanganate oxidizing agent, absorbance in ultraviolet region and
Folin Ciocalteu method (Jasicka-Misiak et al., 2012). None of the method is 100% accurate
for TPC determination not only due to variety of phenolic compounds present in natural
materials but also due to presence of some other substances which can interfere in the
determination of total phenolics present in natural materials.
A study was conducted by Ferreira et al. (2009) to evaluate the total phenolic
contents of honey samples. They compared TPC values of pure honey samples and phenolic
extracts of honey (phenolic contents of honey were prepared by eluting with methanol
through amberlite column). Values for entire honeys and phenolic extracts were 226.16 to
727 and 132.17 to 204.24 mg/kg, respectively which indicated the involvement of
interferences in case of entire honey analysis. Therefore, other compounds like ascorbic acid,
carotene interferes with the total phenolics. Due to large diversity of structures of phenolics
and even lack of availability of many standards and structure for HPLC analysis this is
feasible to determine the total phenolic of honey and other products by Folin-Ciocalteu
method the most popular and commonly known as the total phenolic assay (Hagerman, 2002;
Tsiapara et al., 2009; Dai and Mumper, 2010). Although some limitations are present in
Folin Ciocalteu method but it is well renowned due to its simplicity, reproducibility and
applicability to the wide range of phenolic compounds present in food grains, vegetables,
fruits, juices, honey and other natural products (Silici et al., 2010).
According to Isla et al. (2011) phenolic contents of honey varied with varying floral
source even in the same geographical origin. They analyzed thirteen honey samples from
provinces of northwest Argentina. Multifloral honeys contained higher phenolics in the range
of 875.75 μg GAE/g of honey weight. Phenolic contents of monofloral honeys were
approximately three times lesser than multifloral honeys
2.3.2 Flavonoids
Flavonoids are found to exhibit biological activities like antiulcer, antiarthritic,
antiangiogenic, estrogenic, anticancer, inhibition of prostaglandin synthesis, inhibition of
topoisomerases, anti-allergic activities, anti-inflammatory, antibacterial, antithrombotic and
antioxidant etc. (Ranjbaria et al., 2012; Escuredo et al., 2013 Liu et al., 2013). Solvents like
21
chloroform, benzene and diethyl ether were best for extraction and isolation of flavonoids
aglycone. In case of polar aglycone flavonoids more polar solvents like methanol, ethanol,
Basic structure Flavonoid Quercetin R = OH (Flavonols)
Apigeni, R = H (Flavones) Naringenin R = H (Flavanones)
Genistein R = H (Isoflavones) Pelargonidin R = H (Anthocyanidins)
water, acetone or mixture of solvents can be used. Qurecitin and catechin are used as
standard in most of TFC studies (Bhat et al., 2009). Flavonoids were found to inhibit the
oxidative damage in variety of ways: Flavonoids have ability to trap reactive oxygen species,
scavenge the free radicals, can trap the metals by chelation and can inhibit many enzymes
22
responsible for generation of super oxides (Pyrzynska and Biesaga, 2009). Basic structures of
some dietary flavonoid are given above. Meda et al. (2005) determined total phenolics, total
flavonoids and proline contents of 27 monofloral, multifloral and honey dew honeys. Total
phenolic, flavonoids and proline were determined by Folin ciocaltteu, Dowd and ninhydrin
methods, respectively. Qurecitin R2= (0.999) was used as standard for flavonoid estimation.
Total flavonoids were in the range of 0.17 to 8.35 with mean value of 2.57 mg/100 g.
Correlation between total phenolic and flavonoid contents was very low (r=0.11) while
correlation between flavonoids and antioxidant contents was also very low. Therefore
flavonoids of specific structure can show antioxidant behavior. It is believed that particular
hydroxyl group of flavonoid structure can donate electron or hydrogen atom to free radical.
Candiracci et al. (2012) measured the antifungal activity of flavonoid extracts of honey
against Candidana albicans. Flavonoids were extracted from honey with help of Amberlite
XAD-2 nonionic resin of polystyrene. Extraction of hydrophobic flavonoids was carried out
with the help of diethyl ether and analyzed (Bhat et al., 2009).
2.4 Medicinal properties of honey
Religious texts from different origins reported honey as a great gift for human beings.
Quran the Holy book of Muslims reported the processes of honey production and its
medicicinal importance in the words translated as “And your Lord inspired the bees, saying:
Take your habitations in the mountains and in the trees and in what they erect. Then, eat of
all fruits and follow the ways of your Lord made easy (for you). There comes forth from their
bellies a drink of varying colors wherein is healing for men. Verily in this is indeed a sign for
people who think.” A holy book of Muslims dated back to 8th
century AD reported that Holy
Prophet Mohammad (Peace be upon him) recommended honey for treatment of diarrhea
(Bogdanov et al., 2008).
The Bible quoted in this way “How sweet are your words to my taste, sweeter than
honey to my mouth”. In another place Bible reported honey and milk as foods of Heaven by
saying “And I have come down to deliver them out of the hand of the Egyptians and to bring
them up out of that land unto a good land and a large, unto a land flowing with milk and
honey. Its use is reported even before 4000 years. Tables containing recipes on the use of
honey as dressings were formulated before 2100 B. C. Literature written in 2000 to 1000 B.
23
C. in Egyptian containing 500 recipes on the use of honey as medicine was also discovered.
Literature written by Indian Ayruvedic physicians, Hippocrates and Roman physician on the
use for treatment of inflammation, internal diseases, boils, burns, abscesses, gastrointestinal
disorders, infected and chronic wounds, asthma, burns, cataracts, heart disease, cancer, eye
ailments and skin ulcers is enough to prove the historical importance of honey (Orhan et al.,
2003; Molan, 2006; Ouchemoukh et al., 2007; Ferreira et al., 2009; Werner and Laccourreye,
2011; Fearnley et al., 2012; Kassim et al., 2012; Packer et al., 2012; AL-Waili et al., 2013;
Lazim et al., 2013). Although honey and its products have been used for a long time for
therapeutic and nutritional purposes but research work to explore therapeutic properties of
honey has gain popularity only recently (Lusby et al., 2005; Papadopoulou et al., 2005;
Roussis et al., 2005; Blasa et al.,2007; Berg et al., 2008; Boorn et al., 2010; Atrott et al.,
2012). First scientific effort on the therapeutic importance of honey was published in 1936.
Analysis of honey for therapeutic purposes on scientific grounds leads to commercialization
of many brands of honey for medicinalpurposes in different regions world wide. The heather
honeys, The Medihoney, Activon, L-Mesitran, Honey soft and Manuka honeys were
available in countries like Australia, USA, New Zealand, England, Netherland, Greece and
Belgium for excellent treatment of infections, healing of wounds, inflammation suppression,
epithelium growth and tissue granulation (Molan, 2002a; Simon et al., 2006; Mullai and
Menon, 2007; Werner and Laccourreye, 2011; Robson et al., 2012). Deficiency of honey
brands available as phytomedicine emphasized the need for research of different varieties of
honey to explore new antibacterial aspects and medicinal properties (Voidarou et al., 2011).
Honey due to its antiseptic properties is also used to making face masks and products for
treatment of skin with different types of natural products (Mahindru, 2007). Therefore,
analytical chemists should pay more attention to analyze the chemical aspects of honey
(originated from different sources) which may contribute towards its therapeutic effects.
2.4.1 Antioxidant potential
According to Huang et al. (2005) an inverse correlation was found between
antioxidant fraction (quality and quantity) of food items consumed and occurrence of many
diseases like inflammation, cancer, cardiovascular disease and aging-related disorders.
Therefore, dietary materials rich in antioxidants gained popularity (Amarowicz et al., 2012;
24
Devarajan and Venugopa, 2012; Silva et al., 2013). Complexities of antioxidant composition
of natural foods make it difficult and expensive for a chemist to extract, isolate and analyze
complete antioxidant composition. On the other hand, total antioxidant potential of food
materials may be different from sum of isolated components (Alvarez-Suarez et al., 2012).
Therefore, researchers and nutritionists are always interested to determine the total effective
antioxidant potential of natural food stuffs. Antioxidant activities of any food product may be
assessed by chemical and biochemical methods based on reactions involved in absorption
with λmax. in UV-Vis. region which facilitate these determinations with the help of
spectrophotometer. In vitro antioxidant assays are involved in (1) transfer of hydrogen atoms
or (2) donation of electrons by antioxidant components of reagent to oxidative free radical.
(1) Hydrogen Transfer Assay: Chemistry involved in antioxidant action of hydrogen
transfer antioxidants is given below.
(a) Initiation: Oxidative damage starts with cleavage of bonds followed by formation of free
radicals. Free radical thus generated can react with molecular oxygen to produce peroxy free
radical.
R2X2 2R° + X2
R° + O2 ROO°
ROO° + LH ROOH + L°
(b) Propagation: These peroxy free radicals are highly reactive and facilitate the generation
of free radicals in surrounding environment and can lead to oxidative damage of living tissue.
L° + O2 LOO°
LOO° + LH LOOH + L°
(c) Inhibition: An antioxidant compound can inhibit the oxidative damage by donation of its
hydrogen atom to reactive peroxy free radical
LOO° + AH LOOH + A°
(d) Termination: All produced free radical combine to each other to produce stable system
A° + (n - 1) LOO° nonradical products
LOO° + LOO° nonradical products
(2) Electron transfer assay: These assays are involved in the transfer of electrons from
antioxidant compounds to oxidative compounds. Actual chemistry involved in this reaction
resembles to the chemistry of redox titrations. Electron donors and acceptors are specific and
25
oxygen is not involved in both electron transfer assays and redox titrations. Chemistry of this
reaction is given below
Oxidant compound + e from antioxidant compound reduced oxidant compound +
oxidized antioxidant compound
Ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity
(TEAC), Cu2+
reduction capacity, N, N-dimethyl-p-phenylenediamine (DMPD) and 2, 2-
diphenyl-1-picrylhydrazyl radical scavenging power (DPPH) assays are the examples of
electron transfer assays.
Oxygen radical absorbance capacity (ORAC) assay: This assay is based on the
consumption of fluorescent dye fluorescein which acts as molecular probe. The fluorescence
decay of fluorescein is an indication of damage from its reaction with the peroxyl radical.
DPPH assay in some reactions is also involved in transfer of hydrogen while in other it might
involve in electron transfer from antioxidant to DPPH radical (Foti et al., 2004; Alvarez-
Suarez et al., 2012; Tenore et al., 2012; Gorjanovic et al., 2013; Tuberoso et al., 2013).
DPPH assay which involved in production of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free
radical of violet color structure is given below.
2, 2-diphenyl-1-picrylhydrazyl (DPPH)
Antioxidants present in materials can donate an electron or hydrogen atom to this free
radical. This can be estimated by degradation of violet color of this radical at 517 nm with
the help of spectrophotometer. FRAP assay which is involved in the reduction of ferric to
ferrous ion lead to color changes which are quantified with spectrophotometer at 700 nm.
Some assays are also developed to measure the scavenging power of some other ions
and radicals like hydrogen peroxide scavenging power assay, peroxide radical scavenging
assay, superoxide anion radical scavenging assay, singlet oxygen scavenging activity and
26
peroxynitrite scavenging activity (Martinez-Tome et al., 2001; Wright et al., 2001; Squadrito
and Pryor, 2002).
Honey is also found to act against deteriorative, oxidative reactions occurring in
foods due to heat, light, metals and enzymes (Alvarez-Suarez et al., 2012; Tenore et al.,
2012). Honey has ability to inhibit the browning of vegetables and fruits. Many studies have
also menifested that it prevents lipid oxidation of meat and other food items in addition to its
antimicrobial activity. Its antioxidant potential makes honey beneficial for human beings as
healthy food, medicine and as potential preservative for food stuffs (Nagai et al., 2001;
Mundo et al., 2004; Ferreira et al., 2009). It is therefore, important to determine total
antioxidant potential along with qualitative and quantitative analysis of antioxidant
components. DPPH, FRAP, β-carotene bleaching and ABTS assays are most common for
determination of total antioxidant potential of honey (Saxena et al., 2010; Brudzynski and
Miotto, 2011; Isla et al., 2011; Alvarez-Suarez et al., 2012; Tenore et al., 2012).
2.4.2 Antimicrobial properties of honeys
Antimicrobial properties of honey are due to presence of a variety of bioactive
compounds, low moisture contents and acidic behavior. Low water content makes it highly
osmomolar solution of sugars which help to absorb moisture content of microbes and kill the
microbes by dehydration and make it a potential antimicrobial agent (Al-Habsi and Niranjan,
2012; Moussa et al., 2012b). On the other hand, acidic environment of honey also resist the
growth of any microorganism. Extensive study on antimicrobial properties from different
area has confirmed the broad spectrum antimicrobial behavior of this natural medicinal
product. Manyi-Loh et al. (2010) also claimed that honey is excellent antimicrobial agent
due to acidic behavior, osmolarity, non-peroxide scavenging activity and presence of
hydrogen peroxide (Brudzynski et al., 2012).
Two major assays used to evaluate the antimicrobial activity of material are plate
assay and turbidometeric assay. Turbidometeric assay is based on the measurement of
turbidity of test sample with spectrophotometer because direct relationship is present
between absorbance and concentration of microbes. The sample concentration at which
minimum microbial growth is observed is known as minimum inhibitory concentration
(MIC). This method is helpful to determined MIC90, MIC50 etc. In plate assay disks of wicks
27
paper were impregnated with test sample and put on the agar inoculated with microbes. In
this assay inhibitory zones were produced around sample disks (Bhat et al., 2009; Parmar et
al., 2013).
Silici et al. (2008) analyzed antimicrobial potential of fifty Rhodendron honeys from
some locations of Turkey. Rhodendron honeys were also known as poisonous honey. Honey
extracts of different concentrations were prepared in sterilized saline and applied against
eleven bacterial strains (Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis,
Escherichia coli, Listeria monocytogenes, Mycobacterium smegmatis, P. mirabilis, P.
aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Candida albicans,
Saccharomyces cerevisiae and yeast strain of Yersinia enterocolitica. Low concentrations of
10 and 25% were not effective to inhibit the growth of microbes at all while microbes had
shown sensitivity at honey concentrations of more than 50%. Proteus mirabilis and
Pseudomonas aeruginosa were found to be the most sensitive to honey among all tested
microbes, Staphylococcus aureus, Aeromonas hydrophila, Bacillus subtilis, Mycobacterium
smegmatis, Listeria monocytogenes, and Salmonella typhimurium had shown moderate
sensitivity against all studied honey samples while Bacillus cereus and Yersinia
enterocolitica were almost resistant to honey at all concentrations.
Brudzynski and Miotto et al. (2011) analyzed antibacterial activity of honey samples
against two bacterial strains Bacillus subtilis and Escherichia coli. Fresh cultures of bacterial
strains were prepared in Mueller-Hinton Broth. Antibacterial activities were determined by
using serial dilution method in 96 well plates. After incubation of plates at 37°C for 24 hours
absorbence was measured at 595 nm with the help of ELISA 96 well microplate reader.
MIC90 of all analyzed honey samples were developed by using formula given below
Inhibition of Growth (%) = Abs. of Control well – Abs. of sample well/ Abs. of Control well
MIC90 of analyzed honeys were in the range of 6.25 to 50%. Buckwheat honeys and
wildflower honeys were excellent antibacterial agents with very low MIC90. Correlation of
antibacterial activities and total phenolic, color, milliard reaction products and oxygen radical
antioxidant capacity was determined. Maximam correlation R=0.737(P<0.0001) and
R=0.649 (P<0.002) was found between inhibition of E.coli and ORAC and E.coli and
28
maillard rection products (MRLPs), respectively while correlation of E.coli inhibition with
color and total phenolics was in the range of 0.534 to 0.600. On the other hand correlation of
these properties with growth of Bacillus subtilis was low. All analyzed honeyswere found to
exihibit antibacterial and antioxidant potential.
A study conducted to compare the physicochemical and functional properties of
honeys produced by Apis mellifera and meliponinae (Ecudotian stingless bee) reported that
%DPPH radical inhibition and β-carotene inhibition activities of honeys produced by
meliponinae are higher than Apis mellifera. Antibacterial activity values against Gram
negative and Gram positive bacteria are higher for Apis mellifera than meliponinae. Honey
bee species are also responsible for some enzymatic differences in the composition of honey
(Guerrini et al., 2009). Meliponinae forage for flowers as well as for mature fruits therefore,
some properties of honey can vary for different species. Moisture contents of honeys
produced by meliponinae were much higher than honey produced by other honey bees. Some
chemical attributes of honey are dependent on the honey bee specie like sacharase, proline
concentration and activity of glucose oxidase (Hermosin et al., 2003).
In another study by Voidarou et al. (2011) investigated the antimicrobial potential of
60 Greece honeys against Bacillus cereus, Staphylococcus aureus, Escherichia coli,Bacillus
subtilis, Streptococcus pyogenes and Salmonella typhimurium. Honey solutions (0 to 75%
w/v) were analyzed for antibacterial inhibition by well diffusion assay. Zones of inhibiton
produced by different honey samples were in the ranges of 0.90 to13.35, 3.35 to 12.48, 1.85
to 12.35, 0.00 to 11.84 and 4.75 to 12.35 mm for Staphylococcus aureus, Escherichia coli,
Salmonella typhimurium, Streptococcus pyogenes and Bacillus cereus, respectively. This
study found that honey was not only factor responsible for antibacterial activities. In order to
discovery of new medicinal products further research work was proposed to check the
antibacterial potential of honey against different pathogens.
Gulfraz et al. (2010) evaluated the antimicrobial properties of honey samples
collected from beekeepers. They used the agar-well diffusion method to determine the zones
of inhibition produced by different formulations of honey samples against Escherichia coli,
Staphylococcus aureus, Aspergillus niger and Candida albicans. Pure honey samples due to
hyperosmotic effect have shown maximum inhibition of microbes while diluted formulations
29
of honey were also effective to inhibit the microbial growth which may be due to synergistic
effects of hydrogen peroxide, volatile substances, antioxidant fraction and low pH of honey
samples. On the other hand, presence of nondigestible oligosaccharides like fructo-
oligosaccharides, galactooligosaccharides and inuline may act as prebiotics and can enhance
the growth of beneficial bacteria by suppression of growth of harmful bacteria especialy in
the intestine (National Honey Board, 2005). However, to explore the optimum antimicrobial
activity of our honeys it is essential to check the antimicrobial activities of fresh natural
honeys against a panel of microbes with different protocols because volatile components and
peroxide inhibition potential of honey decrease with the passage of time.
2.4.3 Wound healing properties
Wound healing potential of honey provided it a special place for treatment of burns
ulcers, surgical incision sites, traumatic wounds, sloughed wounds and pressure ulcers
(Lusby et al., 2002; Bardy et al., 2008; Jull et al., 2013). According to Moore et al. (2001)
clinical evidence about honey for treatment of superficial burns and wounds was not
considerable. However, Jull et al. (2013) reviewed 2554 participant and concluded that
superficial and partially thin burns can be treated with honey more efficiently than
conventional wound healing dressings. Brolmann et al. (2012) conducted a survey of
literature about healing properties of honey and found 109 strong evidences which support
the use of honey to heal topical burns in very short time. Another clinical study conducted on
the acute, chronic and heavily colonized wounds which failed to cure by ordinary
management were recovered by honey. Lazim et al. (2013) studied the wound healing effect
of Tualang honeys in two groups of post tonsillectomy (operative procedure for tonsillitis)
patients. Healing of wounds caused by operative process is very important because wounds
interfere with patient‟s recovery from surgery. Two groups of patients were treated with
Tualang honey. One group was treated with Tualang honey intraoperatively, orally and other
group was treated with sultamicillin. Healing process was studied after IST
, 3rd
, 7th
and 14th
day of treatment with help of endoscopic photographs. Healing processes was much higher in
patients treated with Tualang honey as compared to other group.Honey was reported to
suppress the malignant wounds in just twenty four hours. It was also found to act against
ulcers and inflammation (Hamzaoglu et al., 2000; Swellam et al., 2003; Pipicelli and Tatti,
30
2009). Many functional properties of honey were found to support its wound healing
properties. Many bacterial species which induce wounds like Staphylococcus aureus,
vancomycin resistant Enterococci, vancomycin sensitive Enterococci, Pseudomonas,
Aeruginosa, enteric bacteria, Stenotrophomonas species, Acinetobacter baumannii
werereported to be inhibited by honey. (Cooper et al, 2002; French et al., 2005; George and
Cutting, 2007; Blair et al., 2009; Henriques et al., 2010; Sherlock et al., 2010; Cooper et al.,
2011; Jenkins et al., 2011). Formation of biofilms enhanced the wound chronicity. Clinicians
are interested to reduce the formation of biofilms to treat the wounds. Application of high
concentration of honey in infected area reduces biofilm formation (Alandejani et al., 2009;
Merckoll et al., 2009; Maddocks et al., 2012). Honey helped in debridement of wound more
efficiently than hydrogel. High levels of proteases in chronic wounds deteriorated the
cytokines, growth factors and extracellular matrix. Osmotic properties of honey enhanced
lymphatic flow which reduced microbial load and stimulates the lymphatic flow in wounded
area (Molan, 2006; Gethin and Cowman, 2009). Antioxidants in honey not only scavenge
reactive oxidative species produced by neutrophils but also induce anti-inflammatory activity
in wound and burns in the area of wound. Honey can also absorb free iron radicals produced
due to breakage of haemoglobin molecules in wound area (Subrahmanyam et al., 2003;
Henriques et al., 2006; Berg et al., 2008; Jull et al., 2013). Medical grade licensed wound
care products containing honey became available first time in 1999 and now such products
are widely used in different forms. First wound care product was irradiated tube of blended
honeys gain regulatory acceptance by the Australian Therapeutic Goods Administration.
Now a range of honey products are available like Activon Tube, Algivon, Activon Tulle,
Actilite, Actibalm, Algivon Plus, Algivon Plus Ribbon and many more (Seckam and Cooper,
2013).
Therefore, honey can provide moist environment to wounds, inhibit microbial growth
and easy to apply which make it excellent dressing for different types of wounds. Progressive
dilution of honey by wound‟s exudates along with the absorption of some of its constituents
into the blood stream in area of wound makes its antiseptic and osmotic effects just for two to
three days.It draws exudate of wound surface and creates a non-adherent moist interface
between wound bed and the dressing. After absorption of wound exudates by honey internal
blood circulation fresh fluid flows (Pipicelli and Tatti, 2009). Antibacterial activity of honey
31
is reduced due to dilution by wound exudates so it is better to provide a secondary dressing
along with honey which enhances the wound healing properties of honey. Application of
sufficient amount of honey and frequent changes of dressings may help to minimize the
effect of exudate in addition it also acts as anti-inflammatory agent. Mixture of honey and
sodium alginate make hydocolloidal sheet which absorbs much higher amount of exudates
and keeps honey in contact with wounded skin (Molan, 2002a; Molan, 2002b; Molan, 2002c;
Molan, 2004). Dressings of mixture of honey and curdled milk were used to treat severe
burns and wound of soldiers in ancient times. Manuka honey and Medihoney gained great
popularity due to effectiveness against inflammation and skin lesions and even more
effective than pharmaceutical preparations (English et al., 2004; Johnson et al., 2005; Simon
et al., 2006).
2.4.4 Antitumor potential
Cancer is a deadly disease which affects the human being worldwide. Although
cancer chemotherapy is available but its 100% eradication has not yet been possible.
Alternative treatment facilities like surgery, targeted therapy and radiation therapy are also
available. But there existed no remedy without side effects (Jaganthan and Mandal, 2009).
On the other hand, 50% of the anticancer drugs in last twenty years were directly or
indirectly originated from botanical sources (Fauzi et al., 2011). The phenolic acids,
flavonoids, ellagic acid, protocatechuic acid, vanillic acid, carotenoids and polyphenolics are
responsible for anticancer properties of honey (Jaganthan and Mandal, 2009; Tsiapara et al.,
2009; Kasim et al., 2010; Saxena et al., 2012). One of the drawbacks of cancer chemotherapy
is the inhibition of drug action by P-glycoproteins of ABC transporter family. These
inhibitors are produced by cancer cells in response to the drug action. P-glycoproteins are
thought to be inhibited by combined action of group of polyphenolic flavonoids like
genistein, querecetin, naringenin, Kaempferol, biochanin and chrysin. Flavonoids have
ability to inhibit the action of ATPases involved in the removal of drug from cells to facilitate
the entry of drug into the cell. Flavonoids are also thought to play a role of substrate for P-
glycoproteins. It is thought to act as a chemo sensitizer for cancer chemotherapy. Research
efforts should be devoted to explore the role of flavonoids as sensitizer in cancer
chemotherapy. In this way honey can replace the expensive synthetic chemo sensitizer
32
(Jaganathan and Mandal, 2009). Many evidences proved the asnticancer properties of honey.
Honey has been proved effective: in laparoscopic surgeries to prevent the implantation of
tumors, in the prevention of chemotherapically induced neutropenia, intermediately effective
against tumors and significantly inhibited the metastatic growth. Honey preparations were
also proved effective in prevention of immune depressant infections in the children captured
by blood cancer. Anti-cancer activities of 5-fluorouracile and cyclo-phosphamide were
significantly enhanced with the addition of honey (Smirnova et al., 2000; Pipicelli and Tatti,
2009).
Kasim et al. (2010) reported the anti-inflammatory activity of Malaysian honey
against different types of cell lines. Phenolic fractions were extracted by eluting acidified
honey solution through XAD-2 column. Sugars and other polar compound were eluted with
distilled water. Phenolic fraction was obtained by elution with methanol. Anti-inflammatory
activity of honey phenolic extracts was evaluated by colorimetric 3-(4, 5-dimethythiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Nitric oxide scavenging activity was
determined by nitric oxide inhibition assay and phenolic profile was evaluated by HPLC and
LCMS analyses. Ellagic acid was the most prominent phenolic acid with mean value of
3295.8 μg/100 g of honey while Kaempferol was in low amount with a mean value of 16.12
μg/100 g. Gallic acid, Chrysin, Luteolin, Querecetin, Chlorogenic acid, Caffeic acid, p-
Coumaric acid, Myrecitin, Hesperitin and Ferulic acid were also determined. Honey extracts
has against shown less reasonable anti-inflammatory activity agon the treated cell lines while
inflammation in cell lines treated with tumor necrosis factor-α (TNF-α) were found to be
inhibited 100% at a concentration of 250 μg/mL of honey extracts. On the other hand high
production of nitric oxide was raised up to 20 times during honey extract treatment as
compared to cell growth which indicated the anti-inflammatory action of honey extract.
Antioxidant activity and phenolic compound were responsible for in vitro anti-inflammatory
activity. Malaysian honeys were found to exhibit varying anti-inflammatory activities.
Although limited work has been reported on the potential of honey so far both in vitro
and in vivo studies are required to explore anticancer potential of honey from different
origins including mechanisms of action.
The literature survey helped to predict that botanical origin, seasonal variation,
geographical location and specie of honey bee involved in honey production collectively
33
determined physicochemical properties, biological composition and medicinal behavior of
honey. In the context of the continuous emergence of antibiotic-resistant pathogens, some
alternative or traditional topical antimicrobials have been reintroduced into modern wound
care, one such example being honey. Therefore, honey is not only a healthy nutrition but also
a therapeutic agent. In addition to these properties, it also acts as a biomarker for
environmental pollution. These aspects made it a promising natural product with medicinal,
nutritional and environment monitoring potential. This made it interesting product for
nutritionists, physicians and analytical chemists for identification and quantification of its
constituents and properties. In view of above comprehensive and critical review current study
was planned to elucidate the physicochemical, antioxidant, antimicrobial and antitumor
potential of honeys from selected areas of Pakistan. Data of our honeys was compared with
international standards of honeys as well as to the data reported for honey samples from
different regions of the world. Another objective of current study was to detect adulteration
in branded and bee keeper‟s honey samples by comparison with adulterated and natural
honey samples. Principal component analysis and cluster analysis were used to classify the
honey samples. This project will be helpful for establishing the baseline information of
Pakistani honeys with regard to their medicinal and chemical properties.
34
CHAPTER 3 MATERIALS AND METHODS
Quantitative and qualitative analysis were performed in laboratories of the
Department of Chemistry and Biochemistry and Central Hi-Tech laboratory, University of
Agriculture, Faisalabad while antimicrobial and antitumor analysis were carried out in
Bioassay Protein Molecular Biology Laboratory, Department of Chemistry and
Biochemistry, University of Agriculture Faisalabad. GC/MS analysis for determination of
carbon isotopic ratio was performed in Isotopic Application Division, Pakistan Institute of
Nuclear Science and Technology (PINSTECH), Islamabad, Pakistan. All experiments were
performed on the basis of reported protocols for analysis of honeys. Some extractions (like
flavonoids) were based on selection of best solvents found in literature for these products. At
the same time peroxide activities and antitumor activities were evaluated by methods
reported for different natural products due to lack of literature in support of honey. All
experiments were performed in triplicates except antitumor activities which have been
replicated 5 times.
3.1 Instruments used
Double beam spectrophotometer (Lambda 25, Perkin Elmer, USA), Rotary shaker (
OS 10, Yellow Line, USA), Centrifuge (Sigma, UK), Sonicator (Transonic T 460/ H Elma,
Germany), Muffle furnace (Controller B 170, Nabertherm), Laminar air flow (Memmert,
Germany), Fume hood (ESCO), Incubator (Memmert, Germany), Vortex mixer (Heidolph
Reax Top D-91 Schwabach), Oven (Memmert, Germany), pH and Electrical conductivity
meter (WTW Inolab multi, 720, Germany), Atomic absorption spectrophotometer (Hitachi
Polarized Zeeman AAS, Z-8200, Japan), Abbe refractometer (Atago RX-5000, Atago,
Japan), Water bath (Memmert, Germany), Analytical balance (AUY 220, Shimadzu, Japan),
ELISA microplate reader (BioTeck, USA) and HPLC (LC-10A, Shimadzu, Japan),
Microscope ( Olympus SZX7, Japan), Autoclave Shimadzu, Japan), Automatic water
distillation Apparatus (Aquarius, Adventic CPW-200, Japan). Delta mass spectrometer from
35
Finnigan MAT, the analyzer system and control unit of GD-150 mass spectrometer from
Varian MAT.
3.2 Materials
All the reagents and chemicals used were of analytical grade. Nutrient broth,
resazurin, agar powder, 2,6-dichlorophenolindo-phenol, ethylene glycol monoethylether,
bradford dye reagent were supplied Oxoid (Hamsphire, UK), amoxicillin and fluconazole
strips from Oxoid (Hamsphire, UK), 2, 2-diphenyl picryl hydrazyl (DPPH), trichloroacetic
acid, hydrogen peroxide, ferric chloride and pottasium ferricynaide, cadmium chloride
hemihydrate and Ninhydrin were supplied by Sigma (Aldrich, Germany). Sodium carbonate,
HPLC grade methanol and ethanol, Folin Ciocalteus reagent, monopotassium phosphate,
dipotassium phosphate, potassium iodide, iodine and mercuric chloride, Formic acid, 2-
propanol, acetic acid, acetone, n-hexane, hydrochloric acid, mercuric chloride, aluminium
chloride, Potato dextrose agar, Sabouraud dextrose liquid medium from Applichem
(Darmstadt, Germany), meta phosphoric acid, Whatmann filter paper no. 1 and no 4.
(Schleicher and Schuell, Germany), 0.45 µm filter paper (Biotech, Germany),
3.3 Standards
Gallic acid, ascorbic acid and qurecetin, bovine serum albumin, amoxicillin and fluconazole
from Sigma (Aldrich, Germany), Cu, Cd, Pb, Co, Cr, Ni, Mn, Fe, Ca, Mg, Zn, L-Proline, β-
carotene from Applichem (Darmstadt, German) Vincristin from Korea United Parm. Inc..,
L- Leucine from Sinochem (China National Chemical)
3.4 Sampling
The literature evidenced that almost no work has been reported on honey from central
and Southern Punjab whereas some work was reported from other parts of Pakistan including
Sindh, Balochistan, Khyber Pakhtunkhwa and northern Punjab. Therefore, central and
southern Punjab was selected for present study. Total forty five natural honey samples were
collected from seven localities of northern Punjab of Pakistan. Half of the samples collected
from Faisalabad were of Apis florea (Dwarf honey bee) while other half samples were of
Apis dorsata (Giant honey bee). Beehives were uncapped with heated knife and were directly
36
Table 3.1: List of honey samples collected from different sources
Source of Sample No. of samples
Abdulhakeem 4
Bahawalpur 7
Dunya Pur 4
Faisalabad 12
Layyah 2
Multan 9
Shorkot 4
Karror 3
Beekeeper‟s Sample 6
Commercial Samples 11
Adulterated Sample 2
Table 3.2: List of brand names and respective abbreviation of commercial honey
samples
Brand name Sample ID
Islamic Shahed Centre 1 Is1
Islamic Shahed Centre 2 Is2
Salman´s Honey Su
Marhaba honey Ma
China Honey Ch
Muqeet Honey Mu
Langanese Honey La
Swat Honey Sw
Young´ Honey Yo
Lifestyle Li
Cooperative Honey H
37
extracted into sterilized containers. Honey samples were filtered with cheese cloth to remove
wax and other debris. Samples were transferred in clean plastic bottles to prevent
contamination. Samples were shifted to lab immediately after extraction and stored at room
temperature. Beekeeper‟s honey samples were purchased from beekeepers of different
localities in Pakistan. At the same time commercial honey samples of different brands were
purchased from local markets of Faisalabad, Pakistan. Two adulterated honey samples were
prepared by feeding bees with sucrose and glucose syrup. Weight of collected samples was
in the range of 500 g to 1000 g. Names and abbreviations of commercial samples are given in
Table 3.2. Abbriviations of commercial samples are used to explain the results of all types of
analysis.
3.5 Physicochemical analysis
3.5.1 Physical analysis
Honey solution 10 g/75 mL in double distilled water was prepared and measured the
pH at room temperature with pH meter (Gomes et al., 2010). Electrical conductivity of honey
solution 20% W/V was determined at room temperature with WTW Inolab Multi 720
electrical conductivity meter (Malika et al., 2005 and Saxena et al., 2010).
Ash contents were determined by taking 5 g honey in porcelain crucible. Each sample
was burned at 550°C in muffle furnace for 8 hours. Ash contents were determined by
subtracting final weight from initial weight.
Moisture percentage was determined with the help refractive index measurement of
each honey sample at 20°C. Moisture (%) was determined by comparing RF values with %
moisture given in the Wedmore table given in annexure-I (AOAC, 1990 and Downey et al.,
2005).
Each honey solution (0.25% W/V) was taken in quartz cuvettes and scanned from
200-800 nm for determination of λ-max.
3.5.2 Total protein contents
38
Bradford assay was used to determine the total protein contents. Honey solution 450
µL (10%W/V) was mixed with 2.5 mL dye solution and Incubated for 10 minutes at room
temperature in dark. Absorbance of blue complex was measured at 595 nm. Bovine serum
albumin (0.125-2 mg/mL) was used as standard (R2=0.991) (Alvarez-Suarez et al., 2010).
3.5.3 Total free amino acid
Cadmium ninhydrin method followed by Alvarez-Suarez et al. (2010) was used with
slight modifications. Ninhydrin 1g was mixed with 100 mL analytical grade ethanol, 12.5 mL
acetic acid and 1 mL of cadmium chloride hemihydrate (1.5 g/mL). Honey solution (0.5%
W/V) 2 mL was mixed with 4 mL of ninhydrin solution. Warmed in water bath at 84°C for 5
minutes and then cooled in ice bath. Absorbance was measured at 507 nm against a blank
containing all reacting components except honey. L- leucine (1.25 - 10 mg/L) was used as
standard (R2=0.968). Total free amino acid contents were expressed as L-leucine equivalent
mg per 100 g of honey.
3.5.4 Total proline
Total proline contents were determined using the method of Meda et al. (2005) with
slight modifications. Honey solution (0.5% W/V) 1mL was mixed with 1mL of 80% formic
acid solution. Added 1mL of 3% ninhydrin solution (in ethylene glycol monoethylether) and
sonicated for 20 minutes. Reaction mixture was transferred in boiling water bath for 15
minutes and lowered the temperature up to 70°C for 10 minutes. Added 5 mL of 2 -propanol
(50%) and allowed to cool at room temperature for 45 minutes. Reaction mixture without
honey solution was used as blank. Absorbance was measured at 510 nm. Proline (0-0.5
µg/ml) was used for calibration curve (R2
= 0.98) (AOAC, 1990), Delta mass spectrometer
from Finnigan MAT, the analyzer system and control unit of GD-150 mass spectrometer
from Varian MAT.
3.5.5 Sugar analysis by HPLC
Sugars were characterized by HPLC following the method of Manzanares et al.
(2011) due to simplicity and rapidity of method. Analytical grade methanol solution (60%)
10 mL was added into accurately weighted (0.5 g) crude honey and sonicated for 15 minutes
39
to get homogenous solution. Solution was filtered by 0.45 µm filter. Filtrate (20 µL) was
injected in HPLC with Rezex RCM-monosaccharide Ca+2
bounded Phenominex column.
Results were determined by differential refractive index detector. Glucose, fructose and
maltose were used as standards. Conditions of HPLC are given in Table 3.3
Table 3.3: Conditions for HPLC
Injection Volume 20 µL
Mode Isocratic
Flow Rate 0.6 L/min
Column Temperature 80°C
Mobile Phase Double distilled H2O
Detector LC gradient
3.5.6 Mineral analysis
Mineral profile was determined by dry ashing method of dos Santos et al. (2008) with
some modifications. Porcelain crucibles were washed with tap water followed by distilled
water. All crucibles were soaked in 20% nitric acid for 24 hours and then washed with plenty
of distilled water. Honey sample 5 g each was weighed in these porcelain crucibles. Put these
samples in muffle furnace at 100°C for 1 hour. Rose the temperature up to 550°C and
allowed to burn for 8 hours. Resultant ash was dissolved in 12.5 mL commercial HCl (50%
solution in double distilled water). Mixture was heated to get homogeneous solution. The
solution thus obtained was filter through whatmann No. 1 to get clear solution. Volume was
made up to 62.5 mL with double distilled water and stored into properly washed plastic
bottles. Metal contents in the prepared samples were analyzed using atomic absorption
spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan) following the conditions
described in AOAC (1990).
40
Table 3.4: Operational conditions employed in the determination of metals by AAS
Elements Wavelength
(nm)
Slit
Width
(nm)
Lamp
Current
(mA)
Burner
Height
(mm)
Oxidant gas
pressure
Flow rate
(kpa)
Fuel gas
pressure
Flow rate
(kpa)
Ca 422.7 0.4 7.5 7.5 160 6
Cd 228.8 1.3 7.5 5.0 160 6
Co 240.7 0.2 12.5 7.5 160 6
Cu 324.8 1.3 7.5 7.5 160 7
Cr 359.3 1.3 7.5 7.5 160 120
Fe 248.3 0.2 10.0 7.5 160 6
Mg 285.2 1.3 7.5 7.5 160 7
Mn 279.6 0.4 7.5 7.5 160 7
Ni 232.0 0.2 10.0 7.5 160 7
Pb 283.3 1.3 7.5 7.5 160 7
Zn 213.9 1.3 10.0 7.5 160 6
Burner head of AAS was standard type for all determinations and flame of air-acetylene was
used.
Selected metals included Calcium (Ca), Cadmium (Cd), Cobalt (Co), Copper (Cu),
Chromium (Cr), Iron (Fe), Magnesium (Mg), Manganese (Mn), Nickel (Ni), Lead (Pb) and
Zinc (Zn). Calibrated standards were prepared from the commercially available stock
41
solution in the form of an aqueous solution (1000 ppm). Highly purified de-ionized water
was used for the preparation of working standards. The instrumental operating conditions for
the said elements are summarized in Table 3.4.
3.6 Isotopic ratio analysis of C12
/C13
3.6.1 Procedure for preparation of CO2 from honey samples
Ten mg honey sample was placed in the platinum boat and ignited with research
grade oxygen at 1000C for 8 minutes. CO and CO2 gases were produced. CO was converted
to CO2 by circulating it over CuO gauze in a furnace at 850C for 4 minutes. CO2 was
purified using -60C slush (A mixture of acetone with liquid nitrogen). Purified CO2 was
analyzed on Isotope ratio mass spectrometer for 13
C measurements.
3.6.2 Description of the mass spectrometer analysis procedure
The mass spectrometer was isotope abundance ratio measurement type with double
gas inlet system and has ion collector for simultaneous collection of major and minor ions of
a gas. It has been developed using the double gas inlet system of Delta mass spectrometer
from Finnigan MAT, the analyzer system and control unit of GD-150 mass spectrometer
from Varian MAT. The operation of inlet system, on-line data acquisition and all other
calculations were controlled through a personnel computer. For this purpose necessary
computer software was developed. CO2 gas after ionization and under mass separation in the
analyzer provided ions with m/e as 44, 45 and 46. Mass 46 is received at minor ion collector
while masses 44 & 45 were simultaneously received at major ion collector. After
amplification of both the signals through a highly precise dual amplifier system the ratio of
minor to major signal was obtained through a Digital Multimeter (DMM) with ratio option
over preset integration time.
Mass spectrometric analysis of CO2 was performed through isotopic masses:
12C
16O
16O,
13C
16O
16O,
12C
16O
18O,
13C
16O
17O,
12C
17O
17O and
12C
16O
17O. For
13C analysis,
mass 45 was measured versus combined (mass 44 + mass 46) in CO2. Similarly, for the
analysis of 18
O, one measures mass 46 versus the combined mass of 44 + 45 in CO2. The
isotopic ratio actually measured corresponded to that of all species having mass 46 to all
42
species having mass 44 and 45 which are collected together while measuring 18
O/16
O ratio.
Similar was the case of 13
C analysis.
3.7 Bioactive contents
3.7.1 Total phenolic contents (TPC)
Standard Folin-Ciocaltue method followed by Saxena et al. (2010) and Al et al.
(2009) was used with some modifications to determine the phenolic contents. Honey solution
1mL (10% w/v) was mixed with 5 mL of 0.2 N Folin Ciocalteu reagent mixed well for 10
minutes on rotary shaker. Sodium carbonate 4 mL (75 g/L) was added and incubated for 2
hours. Absorbance of reaction mixture was measured at 760 nm. Methanol was used as
blank. Gallic acid (12.5-200 mg/L) was used as standard (R2
= 0.99). The collected data was
presented as Gallic acid equivalent TPC in mg per 100 g of honey.
3.7.2 Total flavonoids contents (TFC)
Flavonoid extraction from honey was performed in three solvent systems; water,
absolute ethanol and ethanol: water (50:50). Honey 2 g was dissolved in 10 mL of solvent
and transferred on orbital shaker for 60 minutes. Extracts were filtered through Whatmann
No. 1 filter paper. Total flavonoid contents were determined by Dow method followed by
Meda et al. (2005) and Vit et al. (2009) with some modifications. Flavonoid extract 5 mL
was mixed with 5 mL of methanolic solution of AlCl3. Incubate the reaction mixture for 20
minutes 37°C. Absorbance was measured at 410 nm against respective honey extracts as
blank. Qurecetin (0 to 80 mg/L) was used for calibration curve (R2=0.987).
3.7.3 Total antioxidant contents (TAC)
Total antioxidant contents were determined following the method of Saxena et al.
(2010) with slight modifications. Methanolic honey solution 3 mL (4 g/10 mL) mixed with
1.5 mL methanolic DPPH solution (0.02 mg/mL). Reaction mixture was incubated for 15
minutes at room temperature. Absorbance as determined at 517 nm against methanol blank.
Ascorbic acid (R2= 0.99) 0-16 µg/mL and Qurecetin (R
2=0.99) 0-16 µg/mL were used as
standards. Results are expressed as ascorbic acid equivalent (AEAC) and qurecetin
equivalent antioxidant contents (QEAC). Experiment was performed in triplicate
43
3.7.4 Vitamin C. contents
Method of Ferreira et al. (2009) with slight modifications was used for determination
of vitamin C. Crude honey 100 mg was mixed with 10 mL of 1% metaphosphoric acid.
Mixture was sonicated for 60 minutes at 25°C for efficient extraction of vitamin C. Mixture
was filtered through Whatmann No.1. Filtrate (1mL) was mixed with 2, 6-
dichlorophenolindo-phenol (9 mL). Absorbance was measured at 515 nm. L-ascorbic (0 to
0.12 mg/mL) was used for calibration curve (R2=0.98).
3.7.5 Total carotenoid (TCC) and Lycopene contents (TLC)
Carotenoid load in honey was determined by spectrochemical method of Alvarez-
Suarez et al. (2010) with some modifications. Honey solution 10 mL (1g/10 mL) in n-
hexane-acetone (6:4) was centrifuged at 1000 rpm for 10 minutes. Supernatant was filtered
with Whatmann No. 4 filter paper. Absorbance was measured at 450 nm against n-hexane-
acetone (6:4) as blank. Β-carotene 0-0.1 µg/mL was used to draw calibration curve. Another
spectrophotometric method reported by Ferreira et al. (2009) for determination of
carotenoids and lycopene was also used for determination of carotenoids and lycopene.
Carotenoid contents evaluated by both methods were compared to check efficiency of
method. Crude honey 100 mg was mixed with 10 mL acetone-n-hexane solution (4:6).
Mixture was sonicated for 30 minutes to extract carotenoid and lycopene. Organic layer was
separated and filtered through Whatmann No. 1 filter paper. Absorbance was measured at
453, 505 and 663 nm against solvent without honey as blank. Lycopene and carotenoid
contents were determined by the following formulae
Lycopene mg/100 mL = -0.0458A663+ 0.372A505 -0.0806A453
β-Carotene mg/100 mL = 0.216A663- 0.304A505 + 0.452 A453
The results thus obtained were calculated to express mg carotenoids and lycopene contents
per kg of honey.
44
3.8 Antioxidant activities
3.8.1 DPPH radical scavenging activity (RSA)
DPPH RSA was measured by method of Meda et al. (2005) with slight modifications.
Honey solutions and DPPH solution used in section 2.3.1 were mixed 2:1 proportion,
respectively. Reaction mixture was incubated at room temperature for 15 minutes and
measured absorbance at 517 nm against methanol blank. DPPH (0.5 mL) solution mixed with
2.5 mL methanol was used as negative control (Ac) while ascorbic acid was used as standard.
Each experiment was performed in triplicates. Inhibition (%) of DPPH radical was
determined by following formula
Inhibition (%) = [Ac - absorbance of sample or standard/ Ac] × 100
3.8.2 Ferric reducing antioxidant power (FRAP) assay
Reducing power of honey was determined by following the method of Saxena et al.
(2010) with some modifications. Honey solution 5 mL (10% W/V) in ethanol was mixed
with 5 mL of 0.20 m phosphate buffer (pH 6.6). Above reaction mixture was mixed with 5
mL of potassium ferricyanide (1%) and incubated for 20 minutes at 50°C. Trichloroacetic
acid (10%) 5 mL was added and vortexes until constant mixing. Reaction mixture thus
obtained was centrifuged for 10 minutes at 3000 rpm. Supernatant 2.5 mL was mixed with 5
mL of double distilled water and 0.5mL of FeCl3 (0.1%) solution. Absorbance was measured
at 700 nm. Ascorbic acid (0-10 mg/mL) was used as reference standard (R2=0.95).
3.8.3 Peroxide scavenging activity
Peroxide radical scavenging activity was determined by method of Olayinka and
Anthony (2010) with some modifications. Honey solution 8 mL (0.05 g/mL) in deionized
water mixed with 1 mL 40 mM H2O2 in 0.1 m (pH 7.4) phosphate buffer. Samples were
incubated for 10 minutes and measured absorbance at 230 nm against phosphate buffer as
blank while H2O2 in 0.1 m (pH 7.4) phosphate buffer was used as negative control and
ascorbic acid as positive control. Inhibition (%) of peroxide was determined by following
formula
45
Inhibition (%) = [Abs. of –ve control - absorbance of sample or standard/ Abs. of –ve
control] × 100
3.9 Antimicrobial activities
3.9.1 Antibacterial activity
3.9.1.1 Bacterial strains
The Staphylococcus aureus, Bacillus subtilus, Escherichia coli, Pasteurella
multocida were the bacterial strains used in the study for antibacterial activity.
3.9.1.2 Preparation of fresh bacterial culture
Method of Brudzynski and Kim (2011) with some modification was used to prepare
fresh culture of all bacteria used in the study. Nutrient broth (Oxoid, UK) 100 mL was
prepared in Erlenmeyer flask contained glass beads. Buffer solution was added to adjust the
pH 7.4 and autoclaved at 121°C for 15 minutes. Mixture was allowed to cool in laminar air
flow. Broth was inoculated with 100 uL bacteria form stored bacterial culture. Growth
medium was incubated in shaker at 37°C for 24 hours to get 5 × 109
cells per mL.
3.9.1.3 Plate assay/Disc diffusion method
Nutrient agar was prepared by mixing agar medium in distilled water. Agar medium
and 10 mm discs of wicks paper were sterilized in autoclave and allowed to cool in laminar
air flow. Cool agar medium was inoculated with 50 µl fresh bacterial culture. Sterilized discs
were poured with honey and spread in petri plates with positive control in the Centre.
Incubate the petri dishes at 37°C for 24 hours. Zones were measured with zone reader.
Experiment was performed against four bacterial strains (Patton et al., 2006).
3.9.1.4 MIC againt bacterial strains
Minimum Inhibitory Concentrations (MICs) were developed by spectrophotometric
assay using the method of Patton et al. (2006) and Manyi-Loh et al. (2010) with some
modification. Nutrient broth pH 7.4 was prepared and autoclaved. MICs were established in
round bottom 96 well micro plates. Wells of row one from A-F of plate was poured with 100
µL honey while G and H rows of 1st column were poured with water (as negative control)
46
and standard amoxicillin (as positive control), respectively. All wells except 1st row were
poured with 50 µL broth medium. Dilute each row by shifting 50 µL from 1st to 12th well
and discard 50 µL from last well. Pour 10 µL of bacterial solution in all wells. Incubate for
24 hour at 37°C. Poured rezasurin solution (conc.) in each well and absorbance was
measured at 520 nm with the help of ELISA microplate reader. MIC of all honey samples
were evaluated by following formula (Brudzynski and Kim, 2011).
Inhibition (%) = (1- Ab.1/ Ab.2)
Ab.1 = Absorbance of sample or +ve control, Ab.2= Absorbance of -ve control
3.9.2 Antifungal activity
3.9.2.1 Fungal strains
Aspergillus niger, Aspegilus flavus, Aspergillus parasiticus, Trichoderma hanzianum,
Helmenthus spermium, Fusarium solani, Penicillium notatum
3.9.2.2 Preparation of fresh fungal culture
Method of Brudzynski and Kim (2011) with some modifications was used for
preparation of fresh fungal strains. Fresh strains of all studied fungal species were prepared
in sabouraud dextrose liquid medium. Broth was inoculated with 100 µl fungus from stock
culture. Inoculated growth media was incubated in shaker at 28°C for 48 hours to get
approximately 106 cfu/mL spores. Medium was sterilized before inoculation in autoclave.
3.9.2.3 Plate assay
Zones of inhibition were evaluated by disc diffusion method of Patton et al. (2006)
with some modification. Potato dextrose agar medium was inoculated with 50µl from fresh
fungal culture. Thin film of inoculated nutrient agar was poured in petri plates. Sterilized
discs were poured with honey and placed in petri dishes with positive control in the centre.
Petri plates were incubated at 28°C for 48 hours. Zones were measured with zone reader.
Experiment was performed for all fungal strains. Agar medium and discs of wicks paper
were sterilized in autoclave at 121°C for 15 minutes before use and petri plates were
sterilized in oven at 120°C for 12 hours.
47
3.9.2.4 MIC against fungal strains
Minimum Inhibitory Concentrations (MICs) were developed by spectrophotometric
assay using the method of Patton et al. (2006) and Manyi-Loh et al. (2010) with some
modification as given for bacterial strains. Sabouraud dextrose liquid medium broth was used
for fungal strains. Plates were incubated for 48 hour at 28°C and absorbance was measured at
520 nm in ELISA micro plate reader. MIC50 values of all honey samples were evaluated by
following formula (Brudzynski et al., 2012)
% Inhibition = (1- Ab.1/ Ab.2)
Ab.1 = Absorbance of sample or (+ve control), Ab.2= Absorbance of -ve control
3.10 Potato disc assay
Agrobacterium tumefaciens was obtained from Nuclear Institute of Biotechnology
and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Red skinned fresh healthy potatoes
were purchased from local market of Faisalabad, Pakistan. Fresh culture of Agrobacterium
tumefaciens was prepared by inoculation of 100 mL (1.3%) autoclaved nutrient broth pH 7.4
with 10 µL of stock culture. This media was left at 28°C for 48 hours to get 5 × 109
cells/
mL. Spores were counted from Department of Microbiology, University of Agriculture
Faisalabad, Pakistan.
3.10.1 Preparation of honey solutions
Honey solutions (80% V/V) were prepared in sterilized double distilled water. 1000
µl of each honey solution was mixed with 150 µL freshly prepared Agrobacterium
tumefaciens.
3.10.2 Antitumor activities
Antitumor activity was evaluated by procedure followed by (Lellau and Liebezeit,
2003 and Hussain et al., 2007) with some modifications. Red skinned fresh healthy potatoes
were purchased from local market of Faisalabad, Pakistan.Potatoes were surface sterilized for
20 minutes with 20% HgCl2 solution and cut (5 × 5) mm by sterilized cork borer. Seven
potato disks along with positive and negative controls in the center were placed in autoclaved
48
petri dish containing 1.5% agar medium. Potato disks were inoculated with 50 µL of above
honey solutions. All experimental work was done in laminar air flow. Petri plates were
incubated at 28°C for 21 days and tumors were counted under microscope. Each sample was
replicated for five times. % inhibition was determined by following formula
Inhibition (%) = [1- No. of tumors in sample/No of tumors in negative control] × 100
3.11 Statistical analysis
Quantitative analysis of physicochemical, antioxidant and bioactive properties of
honeys was expressed as means ± Standard deviation. Bar graphs were constructed for
comparative study. Analysis of variance and correlation analysis were developed at
appropriate conditions. Principal component analysis and cluster analysis was applied on the
physicochemical properties to classify various honey samples: natural (from different
locations), commercial and beekeeper‟s honey samples in order to select the physicochemical
variables capable for characterization of selected honey samples.
49
CHAPTER 4 RESULTS AND DISCUSSION
Physicochemical properties, bioactive composition, antioxidant profile, antimicrobial
activities and antitumor potential of natural and commercial Pakistani honeys were
determined. Fourty five natural honeys collected from eight localities of central and
Southeren Punjab, Pakistan were analyzed. Mean of all samples from all localities were
compared with the help of bar graphs. Comparison was made for mean of all natural samples
with those of commercial samples; likewise samples of bee fed with artificial sweeteners
were also compared with commercial and natural samples. Honey samples produced by the
Apis florea (dwarf honeybee) vs those of the Apis dorsata (giant honeybee) from the same
locality were also studied.
4.1 Physicochemical properties of honeys
4.1.1 Electrical conductivity (EC)
Electrical conductivity values of natural Pakistani honeys as given in Fig. 4.1 were
in the range of 0.42±0.13 to 0.86±0.02 mS/cm with overall mean of 0.75±0.16 mS/cm. Of all
the analyzed samples mean of EC for honeys from Bahawalpur, Dunya pur, Faisalabad,
Karror, Layyah and Multan were greater than 0.5 mS/cm whereas those for honeys from the
Shorkot were the lowest. Fig 4.2 exhibited the mean of EC values for commercial,
beekeepers, natural and adulterated honey samples where most of commercial samples have
less EC as compared to those of natural samples whereas beekeeper‟s honey samples have
close EC values (0.49±0.00 to 0.77±0.01) to those for adulterated honeys. At the same time
branded honeys have less EC values as compared to natural honeys. However, EC of all
analyzed Pakistani honeys were under the permissible limits of international standards for
honey.
50
Fig. 4.1: Electrical conductivity values of natutal honeys from different locations
EC values exhibited by Uruguayan (0.41±0.08 to 0.99±0.30 mS/cm) (Corbella and
Cozzolino, 2006), Indian (0.33±0.01-0.94±0.01mS /cm) (Saxena et al., 2010) and Moroccan
honeys (0.215 to 0.780 mS/cm) (Malika et al., 2005) honey samples were in close agreement
to our natural honeys. On the other hand EC values reported for honey samples from Portugal
(0.19 ± 0.01 to 0.53 ± 0.01 mS/cm) (Gomes et al., 2010), Irish (0.11 to 0.48 mS/cm)
(Downey et al., 2005) and Argentinian honeys (0.121±0.006 to 0.684±0.007mS/cm) (Isla et
al., 2011) were less than that of our natural honeys but comparable to most of our
commercial and branded honeys. According to a study on Spanish honeys the EC values
(0.2157±0.6790 to 0.5299±0.1106 mS/cm) (Serrano et al., 2004) were less than those for our
natural honey but comparable to our commercial honeys while in another study (Manzanares
et al., 2011) Spanish blossom honeys revealed (0.47±0.17 mS/cm) which was closer to our
commercial honeys but less than our natural honeys whereas honeydew honeys exhibited
(1.21± 0.33 mS/cm) which was much higher than our findings. European Economic
Community (2002) and Manzanares et al. (2011) reported that blossom honeys exhibited less
than 0.80 mS/cm while honeydew honeys exhibited more than 0.80 mS/cm EC. Therefore,
conclusively, on the basis of EC values most of analyzed Pakistani honeys are similar to
blossom honeys except the samples from Layyah which resembles to honeydew honeys.
0.58
0.83 0.78
0.86 0.86 0.82
0.42
0.80
0
0.2
0.4
0.6
0.8
1
1.2
EC
(m
S/cm
)
Sampling Areas
51
0.59
0.86
0.00
0.20
0.40
0.60
0.80
1.00
1.20
D G
EC (
mS/
cm)
Honey bee Spcies
Fig. 4.2: Electrical conductivity values for adulterated, natural and commercial honeys
Fig. 4.3: Mean electrical conductivity values for honeys produced by Apis florea (D) and
Apis dorsata (G)
0.49
0.75
0.49 0.50 0.48
0.77
0.52 0.54
0.25
0.16 0.20 0.21
0.11
0.31
0.42
0.27 0.24 0.24
0.19
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1EC
(m
S/cm
)
Commercial Samples
52
EC values of honey from same geographical origin varied with the honey bee species.
In our study EC values for Apis dorsata was found higher than that of Apis florea as shown
in Fig. 4.3. EC (0.48 ± 0.06 mS/cm) value reported by Guerrini et al. (2009) for honeys
produced by maliponinae (stingless bee) were less than our honeys produced by Apis dorsata
and Apis florae. EC values for honeys produced by Apis mellifera (Malika et al., 2005) were
also very close to those found for our honeys produced by Apis dorsata and Apis florea.The
EC values of Pakistani honeys were found closer to those of international data.
4.1.2 pH
Natural honeys collected from different localities exhibited pH values closer to each
other in the range of 3.81±0.01 to 3.87±0.06 (˂4.0) as shown in Fig 4.4. All honeys collected
from different locations were very close. Values of pH for our commercial sample were also
very close to those of the natural honeys (Fig 4.5). Range of pH values for commercial honey
samples is 3.61±0.02 to 4.24±0.03. So all types of Pakistani honeys analyzed were found to
be acidic in nature.
Fig. 4.4: pH Values for natural honeys collected from different locations
In an earlier study conducted by Kamal et al. (2002) on some floral honeys of
Islamabad (Pakistan), pH values (3.330±0.06 to 6.305±0.10) were greater than those of our
honeys. pH values of our natural Pakistani honeys were closer to the values found for
3.83
3.81
3.81
3.86 3.87
3.81 3.83
3.83
3.65
3.7
3.75
3.8
3.85
3.9
3.95
pH
Sampling Area
53
Portugal (3.7±0.3 to 4.3±0.7) (Gomes et al., 2010) and Italian (3.21 to 3.92) (Spano et al.,
2008) honeys while pH values of our commercial Pakistani honeys were comparable to the
Australian (4.02±0.01 to 4.69±0.01) (Ajlouni and Sujirapinyokul, 2010) and Moroccan (4.00
to 4.42) (Malika et al., 2005) honeys. On the other hand, our data obtained both for
commercial and natural honeys were comparable to Argentinian (3.60±0.2 to 4.10±0.6)
(Baroni et al., 2009), Venezulean (3.30±0.4 to 4.3±0.4) (Rodriguez et al., 2004), Czech
Republic (3.16 to 4.76) (Celechovska and Vorlova, 2001), natural Indian (3.24 to 4.48) (Bath
and Singh, 1999), Uruguayan (3.0±0.40 to 4.3±0.9) (Corbella and Cozolino, 2006), Irish
(3.75-4.61) (Downey et al., 2005) and Commercial Indian honeys (3.7±0.001- 4.4±0.01)
(Saxena et al., 2010). Georgian (6.34 to 6.94) (Taormina et al., 2001), Indian (3.8±0.10 to
5±0.08) (Ahmed et al. 2007) and some Spanish (4.02±0.19 to 4.11± 0.23) (Serrano et al.,
2004) were less acidic than that of our honeys. Acidic behavior of honey is due to certain
metal ions, phenolics, amino acids and gluconic acid whereby their presence and
concentrations depend on floral origin of honey. Presence of Glucose oxidase in honey is
responsible for gluconic acid and peroxide inhibition of honey (Malika et al., 2005). Presence
of glucose oxidase in our honeys was confirmed by excellent peroxide activities. Hence, all
of our honey samples were acidic in nature. Manzanares et al. (2011) found that pH values of
honeydew and blossom honeys were 4.58±0.69 and 3.96±0.28 respectively, thus our honey
samples can be classified as blossom honeys on this scale.
Our honeys produced by Apis dorsata and Apis florea have also very close pH values (Fig.
4.6). Acidic behavior of Pakistani honeys was not only indication of its excellent
antimicrobial action but also good healing agent because acidic honeys help in rapid oxygen
release from haemoglobin (Malika et al., 2005).
54
3.97
3.83
4.04
3.92
4.02
3.61
3.78 3.71
3.94 3.94
3.94
3.88
3.67
4.24
4.01 3.95
3.84
3.96
3.84
3.2
3.4
3.6
3.8
4
4.2
4.4
pH
Commercial Samples
3.88 3.86
3.55
3.60
3.65
3.70
3.75
3.80
3.85
3.90
3.95
4.00
4.05
D G
p
H
Honey bee Species
Fig. 4.5: pH values of analyzed commercial Pakistani honeys
Fig. 4.6: pH values of honeys produced by Apis dorsata (G) and Apis florea (D)
4.1.3 Ash contents
55
0.30%
0.36% 0.30%
0.33%
0.80%
0.31% 0.25%
0.23%
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.01
a
sh (
%)
Sampling Area
Natural Pakistani honeys contained 0.23±0.001 to 0.80±0.001% ash as shown in Fig
4. 7. Among all natural honeys, maximum ash contents (0.80%) were found for honeys from
Layyah and minimum for honeys from Shorkot (0.25%) and Karror (0.23%). Our
commercial honey samples were found to contain less amount of ash as compared to their
natural counterparts. Results for ash contents of all the commercial Pakistani honeys were in
the range of 0.10±0.00 to 0.20±0.00% as shown in Fig. 4.8, where %ash for fifteen sample
was 0.10% and 0.20% for only two of them. Mean amount of ash in natural honeys analyzed
in this study was 0.36±0.002%. Therefore, ash % of commercial and natural honey samples
was significantly different.
Fig. 4.7: Ash (%) of natural Pakistani honeys collected from different location
Ash values (0.046±0.00 to 0.774±0.03) of some floral Pakistani honeys in an earlier
study (Kamal et al., 2002) were found very close to those for our honey samples. Our natural
honeys exhibited ash% closer to but commercial honeys contained less than those found for
Turkish (0.20±0.04 to 0.5±0.03%) (Kucuk et al., 2007), Moroccan (0.18 to 0.57%) (Malika
et al., 2005), Venezulean (0.19±0.006 to 0.64±0.0300%) (Rodriguez et al., 2004) and Czech
Republic honeys (0.16 to 0.80) (Celechovska and Vorlova, 2001). Venezuelan honeys in
another study conducted by Vit et al. (2009) were found to contain (0.03±0.00 to 0.13±0.01
with mean value 0.06±0.04) much lower ash contents as compared to our results. Our natural
56
as well commercial honeys contained ash contents comparable to Indian (0.03±0.01 to
0.43±0.04%) (Saxena et al., 2010), Portugal‟s (0.07 ± 0.01 to 0.35 ± 0.2%) (Gomes et al.,
2010), Irish (0.03 to 0.46 %) (Downey et al., 2005) and Argentinean honeys (0.063±0.036%)
(Finola et al., 2007). Argentinian (0.07±0.05 to 0.2±0.1) (Baroni et al., 2009) and some
Indian (0.08 to 0.11) (Ahmed et al., 2007) honeys contained much lower ash contents than
our natural honeys but comparable to our commercial honeys. Ash contents exhibited by
Romanian (0.03±0.01 to 1.23±0.01%) (Al et al., 2009), some Spanish (0.003 to 0.990 %)
(Soria et al. 2004) and French (0.06-0.54 %) (Ouchemoukh et al. 2007) honeys were found
scattered widely around our findings. Average ash (%) for two type of honeys produced by
Apis florae (D) and Apis dorsata (G) collected from same locality (Faisalabad, Pakistan)
were 0.22±0.0004 and 0.33±0.0008, respectively as shown in Fig. 4.9.
Fig. 4.8: Ash (%) of commercial Pakistani honeys
A study conducted by Guerrini et al. (2009) determined ash values for maliponina
(stingless honey bee) (0.28±0.04 g/100 g of honey) which were more than those for honey
samples
.
0.11%
0.36%
0.10%
0.02%
0.20%
0.10% 0.10%
0.10%
0.10%
0.10%
0.10% 0.10%
0.10%
0.10%
0.10%
0.20% 0.20%
0.10%
0.10%
0
0.001
0.002
0.003
0.004
0.005
0.006
A N B1 B2 B3 B4 B5 B6 IS1 IS2 Su Ma Ch Ma La Sw Yo Li H
ash
(%
)
57
produced by Apis florae but less than those for one produced by Apis dorsata. Ash
contents of all honeys analyzed in this study were in close agreement with data for national
and international honeys. Almost all analyzed Pakistani honeys except honey samples of
Layyah can be classified as blossom honeys on the basis of ash contents as found in the case
of EC and pH values.
Fig. 4.9: Ash (%) of honeys produced by Apis florea (D) and Apis dorsata (G)
4.1.4 Color of honey samples
Color of honey samples was determined as Pfund (mm) by correlation with optical
density of honey samples against glycerine blank. Most of natural honey samples were amber
to dark amber. At the same time all beekeepers honey samples, bee fed honey samples and
most of commercial honey samples were light amber. Cooperative honey was amber and
Young´s honey was light amber in color. Detail values of honey colors are given in
Annexure-II. Ferreira et al. (2009) found that lighter honeys contained lower amount of
flavonoids, β-carotene and lycopene while amber and dark amber contained higher amount of
these constituents. In present study, most of natural honeys containing higher amount of
these components were also found darker in color as compared to commercial and
beekeeper‟s honey samples which contained less amount of these bioactive compounds and
were therefore lighter in color.
0.22%
0.33%
0.00%
0.05%
0.10%
0.15%
0.20%
0.25%
0.30%
0.35%
0.40%
0.45%
D G
as
h (
%)
Honey bee Species
58
4.1.5 Moisture contents
Moisture percentage of natural Pakistani honeys analyzed in this study was in the
range of 17.80±0.28 to 19.67±0.81% as shown in the Fig. 4.10. Among all natural samples
moisture % in samples of Layyah was in small amount with a mean value of 17.80±0.28.
Moisture contents in the samples of the Faisalabad, Multan, Dunyapur and Shorkot were
more than 19%. Higher moisture contents were due to the humid environment or might be
due to immature honey extraction (Ajlouni and Sujirapinyokul, 2010). Previously in a study
conducted by Kamal et al. (2002) moisture percentage of our honeys from Islamabad was
found to be in the range of 17.142±0.27 to 17.97±0.42. Although minor differences were
found which might be due to difference in location of samples but both results indicated that
amount of moisture in Pakistani honeys were under permissible moisture values of
international standards of honeys.
Fig. 4.10: Moisture (%) of natural honeys collected from different localities of Pakistan
In case of commercial Pakistani honeys (as shown in Fig. 4.11) moisture contents of
beekeepers honey samples and branded honey samples were in the range of 13.80±1.06 to
18±0.68 and 15.20±1.22 to 20.60±1.58%, respectively. Moisture contents of our beekeeper‟s
honey samples were less as compared to natural honeys while moisture contents of branded
honeys were comparable to those for natural honeys. Moisture percentage of our adulterated
18.95
18.11
19.67
19.03
17.80
19.02 19.10
18.60
15.5
16
16.5
17
17.5
18
18.5
19
19.5
20
20.5
21
M
ois
ture
(%
)
Sampling Areas
59
honeys was 14.60±1.13%. Moisture in beekeeper‟s honey samples was close to the
adulterated honey samples.
Overall all types of our analyzed honey samples exhibited the moisture contents
almost equal to moisture contents of Moroccan (17.80 to 21.80%) (Malika et al., 2005),
Argentinian honeys in two studies (18.40±1.60%) (Finola et al., 2007), (14.1 ± 0.1 to
18.8±0.0%) (Isla et al., 2011), Czech Republic (13.2 to 19.8%) (Celechovska and Vorlova,
2001), Indian (16.5±0.25 to 19.50±0.14%) (Bath and Singh, 1999), Indian honeys in another
study (17.2 to 21.60%) Saxena et al. (2010), Romanian (15.40±0.06 to 20.00±0.12%) (Al et
al., 2009), Venezulean (17.80±0.24 to 20.40±0.06%) (Rodriguez et al., 2004), (17.20±0.00 to
20.20±0.00%) (Vit et al., 2009), Spanish (16.54±1.05 to 16.63±1.17%) (Serrano et al., 2004),
Spanish honeys in another study (13.0 to 18.7%) (Soria et al., 2004), Portugal‟s (15.9 ± 0.1
to 17.2 ± 0.2%) (Gomes et al., 2010), Australian (10.6±0.30 to 17.8±0.37) (Ajlouni and
Sujirapinyokul, 2010), Argentinian (17.40±0.8 to 18.30±0.90) (Baroni et al., 2009) and
Fig. 4.11: Moisture (%) of commercial honey samples native to Pakistan
14.60
18.79 18.00
15.40
13.80
15.40
17.00 17.00
19.20
15.80
18.80
17.20
20.60
17.60 18.00
17.40
18.20
15.20
17.00
0
5
10
15
20
25
A N B1 B2 B3 B4 B5 B6 IS1 IS2 Su Ma Ch Mu La Sw Yo Li H
M
ois
ture
(%
)
60
21.07
19.03
0.00
5.00
10.00
15.00
20.00
25.00
D G
% M
ois
ture
Species of Honey bee
Uruguayan honeys (16.7 to 18.96 %) (Corbella and Cozolino, 2006). Moisture
contents of Italian (17.4±0.02 to 27.70±0.3%) (Spano et al., 2008) honeys were higher than
Pakistani honeys. Moisture level of honey depends not only on the origin but also on the bee
specie. Italian research proved that moisture level in the honeys produced by meliponinae
(stingless honeybee) was higher as compared to honeys produced by other bee species.
Average moisture (%) in Italian honey produced by meliponinae was 34.1±4.34. This may be
due to foraging of meliponinae bees for ripened fruits along with flowers (Guerrini et al.,
2009). The moisture content in case of our honeys produced by Dwarf and Giant honey bees
from same locality were slightly different. Moisture contents of honeys produced by Apis
florea (D) honey bees were slightly higher than those for Apis dorsata (G) honey bee as
shown in Fig. 4.12. Moisture contents of all types of our honeys were in close agreement not
only to international standards but also obey local standards. Less than 20% moisture
contents of studied honeys rendered high quality, low fermentation rate, less spoilage and
good antimicrobial potential.
Fig. 4.12: Moisture (%) of honeys produced by Apis florea (D) and Apis dorsata (G)
honey bees
61
4.1.6 Refractive index
Refractive index our honey was in the range of 1.4845 to 1.4991 as shown in Fig.
4.13. Refractive index values obtained in this study were close to Indian 1.4825 to 1.4933
(Saxena et al., 2010) and Pakistani honeys of Islamabad region (1.4915 –1.4936) (Kamal et
al., 2002). Wedmore table indicating the relationship of moisture contents of honey with
refractive index is given in Annexure-I.
Fig. 4.13: Refractive Index values studied honeys
4.1.7 Total protein
Protein load of natural Pakistani honeys collected from different locations was
0.96±0.25 to 1.70±0.10 g/100 g as shown in the Fig. 4.14. Mean protein value of honey
samples of Karror was 1.66 g/100 g. Minimum value 0.96±0.25 g/100 g for honeys of
Shorkot and maximum value 1.70±0.10 g/100 g protein was found for honeys of Multan
whereas honey samples from Bahawalpur, Dunya pur and Multan were found to contain
higher protein as compared to other samples protein contents. Protein level of natural
Pakistani honeys were much higher as compared to the data reported for different countries
1.4865 1.4876
1.4845 1.4858
1.4886
1.4860
1.4859
1.4872 1.4881
1.4940
1.4991 1.4982
1.4937 1.4941
1.4885
1.4971
1.4898
1.4923
1.4848
1.4926 1.4916
1.4935
1.4908
1.4860
1.4905
1.47
1.475
1.48
1.485
1.49
1.495
1.5
1.505
Ab
du
lhak
e…
Bah
awal
pu
r
Du
nya
Pu
r
Fais
alab
ad
Layy
ah
Mu
ltan
Sho
rko
t
Kar
ror
B1
B2
B3
B4
B5
B6
IS1
IS2 Su Ma
Ch
Mu La Sw Yo Li H
Re
frac
tive
Ind
ex
62
like Indian (0.048±0.001 to 0.23±0.0023%) (Saxena et al. 2010), Turkish (0.06±0.006 to
0.170± 0.009%) (Kucuk et al., 2007), French (0.37±0.0 to 0.940±0.01 g/ 100g)
(Ouchemoukh et al., 2007), Algerian (0.037 to 0.94 g/100g) (Azeredo et al., 2003),
Romanian (0.002 to 0.125%) (Al et al., 2009) and Italian honeys (0.012±0.0143 to
0.092±0.0123%) (Alvarez-Suarez et al., 2010).
Fig. 4.14: Protein contents of natural honeys from different locations
Values of total protein found for commercial Pakistani honeys were given in the Fig.
4.15. Protein contents of commercial Pakistani honeys were less than natural Pakistani
honeys. Protein values for beekeeper‟s honeys were less than detection limits. Protein values
for commercial honeys were in the range of 0.14 to 1.14 g/100 g. Protein values of natural
Pakistani honeys investigated in this study was found higher than those found in previous
studies by different researchers. Comparison of protein content of honey produced by Apis
florae and Apis dorsata honey bees is given in the Fig. 4.16. Mean protein content of Apis
dorsata was 1.31±0.13 and Apis florea was 1.03±0.13 g/100 g.
0.98
1.67 1.64
1.31 1.32
1.70
0.96
1.66
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Pro
tein
(g
/100 g
)
Sampling Areas
63
Fig. 4.15: Protein contents of beekeeper’s and commercial Pakistani honeys
Fig. 4.16: Protein contents of honeys produced by Apis florea (D) and Apis dorsata (G)
4.1.8 Total free amino acids and proline contents
Proline and amino acids contents of natural Pakistani honeys analyzed during this
study are given in Fig. 4.17 as indicated by brown and green bars. Amount of Proline and
0
1.4
0 0 0 0 0 0
0.71
0.4 0.37
0.65
0.14
0.79
1.14
0.45 0.52 0.49
0.31
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Pro
tein
(g/
10
0 g
)
1.03
1.31
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
D G
Pro
tein
(g/
10
0 g
)
Honey bee Species
64
Fig. 4.17: Total free amino acids and total proline contents of honeys from different
locations
amino acids in natural honeys were in the range of 129.30±0.82 to 661.80±4.49 and
199.05±3.40 to 2336.91±45.57 mg/ kg, respectively. Proline values studied honeys were
26.63±18.81% of total free amino acids. Honeys of Bahawalpur and Karror contained 2336.1
and 1726.30 mg/kg amino acids, respectively which were more than all studied honey
samples. Honey samples of Bahawalpur and Layyah contained maximum proline values.
Total free amino acid and proline profile of beekeeper‟s and branded honey samples are
given in the Fig 4.18 and 4.19, respectively. Beekeeper‟s honeys contained 44.0±0.00 to
305±5.20 and 7.00±0.08 to 47.70±0.08 mg/kg free amino acid and proline levels,
respectively while branded honeys contained 87.20±9.3 to 856.70±4.27 and 41.80±0.08 to
149.00±0.99 mg/kg amino acids and proline contents, respectively.
262.31
661.80
474.15 556.30
658.50
462.86
129.30
529.38 421.10
2336.91
1117.10
857.23
1220.00
840.96
199.05
1726.30
0
500
1000
1500
2000
2500
Total Proline
Total FreeAmino Acid
Sampling Areas
Tota
l Pro
line
and
am
ino
aci
d (
mg/
Kg)
65
47.70 39.70 47.70 33.50 27.60
7.00
275.6
166.7 176.1
44
305
10
0
50
100
150
200
250
300
350
B1 B2 B3 B4 B5 B6
ProlineTotal FreeAmino Acid
Tota
l Pro
line
and
am
ino
aci
d (
mg/
Kg)
Beekeeper's Honey Samples
103.00
46.50
149.00
69.30 41.80 55.20
96.50 82.00 71.00 97.40 78.00
856.7
125.61
323.9
531.1
87.2 91.7
434.4
231.1
617.8
451.7
732.8
0
100
200
300
400
500
600
700
800
900
1000
IS1 IS2 Sw Ma Ch Mu La Sw Yo Li H
Proline
Total FreeAmino Acid
Tota
l Pro
line
and
am
ino
aci
d (
mg/
Kg)
Fig. 4.18: Total free aminos acid and proline contents of honeys collected from bee
keepers
Fig. 4.19: Total free amino acids and total proline level of some branded commercial
Pakistani honeys
66
Total proline contents exhibited by Burkina Fasan (437.80±23.00 to 2169.4±18.40
mg/kg) (Meda et al., 2005) were more than our findings while our natural honeys contained
total proline comparable to those found for honeys of many countries like French (202±13 to
680±17 mg/g) (Ouchemoukh et al., 2007), Argentinian (322±145 to 369±124 mg/kg) and
Spanish honeys determined in two studies (185.4431±126.5551 to 429.53±285.05 mg/kg)
and (800-850 mg/kg) (Serrano et al., 2004; Paramas et al., 2006). Our beekeeper‟s and
commercial honey samples contained much less amount of proline as compared to our
natural as well as international data reported by different scientists. Overall proline contents
in our honeys were 26.63±18.81% of total free amino acids which is similar to studies
conducted on Estonian and Spanish honeys in which major amino acid was proline
(Herminson et al., 2003; Paramas et al., 2006; Rebane and Herodes, 2010). Honeydew
honeys and blossom honeys were also reported to contain significantly different amount of
proline (1069±284 and 602±140 mg/kg respectively) (Manzanares et al., 2011). Minimum
permitted value of proline is 180 mg/kg. This value is indicator of honey ripeness and to
some extent its purity (Manzanares et al., 2011). All our honeys were found to contain
proline more than permitted levels and resembles to blossom honeys. Total free amino acid
contents (153±29 to 1464±123 mg /kg) of Italian honeys (Alvarez-Suarez et al., 2010) were
less than those for natural but more than commercial and beekeeper‟s Pakistani honey
samples.
Comparison of amino acid and proline level of honeys produced by Apis florea and
Apis dorsata in same locality is given in the Fig. 4.20.Total free amino acid and proline load
of Apis dorsata were much higher as compared to Apis florae as shown by brown bars in Fig.
4.20. Overall total protein, total amino acid and proline contents of natural Pakistani honeys
analyzed in this study proved that all those honeys were genuine.
67
27.74
32.48
36.05 32.85
25.90
31.90
27.75
37.33
17.27 19.80
21.79 19.25
15.69
19.83 16.59
22.55
4.64 6.45 6.71 6.63
4.11 5.63 4.52
7.25
0
5
10
15
20
25
30
35
40
45 Fructose
Glucose
Maltose
Fru
cto
se, g
luco
se a
nd
mal
tose
(g /
10
0 g
)
Sampling Areas
Fig. 4.20: Total free amino acids and total proline contents of honeys produced by Apis
florea (D) and Apis dorsata (G)
4.1.9 Sugar profile
Fructose, glucose and maltose contents are shown as brown, green and purple bars,
respectively in Figs. 4.21, 4.22, 4.23 and 4.24. Sugar profile of Pakistani honeys extracted
Fig. 4.21: Sugars profile of natural honeys from different locations
249.3
370.83
526.3
857.23
0
200
400
600
800
1000
1200
Proline Total FreeAmino Acid
D
GTo
tal P
rolin
e an
d a
min
o a
cid
(m
g/K
g)
68
11.10
31.50
26.55
11.77 10.44
25.56
18.50
26.86
12.26
19.09
21.81
13.24 11.28
23.50
18.27
25.26
3.64 5.74
9.42
0.16
7.13
4.40
7.98 9.55
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
A N B1 B2 B3 B4 B5 B6
Fructose
Glucose
Maltose
Beekeeper's Honey Samples
Fru
cto
se, g
luco
se a
nd
mal
tose
g /
10
0 g
From different localities is given in the Fig. 4.21. Amongst all sugars, fructose (25.5±0.25 to
37.33±1.77 g/100 g) was the highest in all natural Pakistani honeys followed by glucose
(15.69±0.12 to 22.25±1.11 g/100 g) as second major sugar and maltose (4.11±0.22 to
7.25±0.52 g/100 g) as third most abundant monosaccharide. Sugar profile of honeys
collected from beekeepers is given in the figure 4.22. Fructose, glucose and maltose values
were 10.44± 0.52 to 26.86± 1.34, 11.28± 0.56 to 25.26±1.26 and 0.16± 0.01 to 9.55± 0.48
g/100 g, respectively. Sugar profile of beekeeper‟s honey samples resembles to the sugar
profile of the adulterated honey which is shown as bar A in the Fig. 4.22.
Fig. 4.22: Sugar profile of commercial honeys collected from beekeepers
Mean of natural samples is shown as N bar in the Fig. 4.22. Most of beekeepers
honey samples have sugar profile different from the sugar profile of natural honeys. Frucose,
glucose and maltose constituents of branded Pakisani honeys were versatile for each honey
sample as shown in the Fig. 4.23. Sugar profiles of Is1 and Is2 (Islamic Shehed1 and 2) and
Li (Life style) honey are similar to sugar profile of natural honey samples. Fructose level of
Ma (Marhaba), Mu (Muqeet) and C (China) honey are higher as compared to natural honeys.
69
Literature survey revealed that Romanian (21.65±0.04 to 42.19±0.02) (20.84±0.02 to
45.79±0.029) (Al et al., 2009), Argentinean (41.1±4.8) (31.70±4.6) (Finola et al., 2007),
Venezuelan (29.2 to 38.7) (29.2 to 38.7) (Rodriguez et al., 2004) and Spanish (33.98±8.81 to
34.72±9.09%) (27.87±7.03 to 33.29±1.96) (Serrano et al., 2004) contained fructose and
glucose, respectively. Maltose contents of Romanian and Spanish honeys were 0.77-5.06 (Al
et al., 2009) and 5.52±0.89 respectively (Serrano et al., 2004). Fructose contents of Spanish
and Argentanian honeys were in close agreement to those found for natural Pakistani honeys
analyzed in this study. On the other hand, Romanian honeys contained a wide range of
fructose as compared to natural Pakistani honeys analyzed in this study.
Fig. 4.23: Sugar profile of commercial honeys of local brands of Pakistan
In case of glucose contents, natural Pakistani honeys contained much less glucose as
compared to glucose content found for honey samples of other countries. However, upper
limit of glucose contents obtained for Pakistani honeys is close or equal to the lower limits of
international data. Fructose glucose ratio is more important quality parameter for
authenticity.
11.10
31.50
27.92 29.25
0.00
44.17
41.04
33.31
20.11
20.65
38.58
27.02
36.47
12.26
19.09 18.76 17.89
0.00
26.61 23.77
19.42
12.08 12.35
22.92
16.46
21.99
3.64 5.74
3.79
8.75
0.06 1.13
5.13 3.58
2.94 5.21 4.21 6.03
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
A N IS1 IS2 Su Ma C Mu La Sw Y Li H
Fructose
Glucose
Maltose
Honey Samples of different brands
Fru
cto
se, g
luco
se a
nd
mal
tose
g /
10
0 g
70
Fig. 4.24: Sugar profile of commercial honeys produced by Apis florea (D) and Apis
dorsata (G)
Pakistani honeys analyzed in present study found to have F/G ratios similar to Romanian
(0.81 to 1.57) (Al et al., 2009), Spanish (1.19±0.08 to 1.28±0.13) (Soria et al. (2004) and
Venezulean (1.19 to 1.39) (Rodriguez et al., 2004) honeys. Sugar profile of natural Pakistani
honeys is similar to the Spanish, Argentanian and Venezulean honeys.
Fructose glucose ratio (F/G) ratios are more important quality parameters according
to Codex Alimentarius standards. F/G ratio more than 1.14 is recomended for non labeled
natural honey sample. All natural honey samples analyzed in our study have excellent F/G as
shown in Table 4.1. Honeys from Abdulhakeem, Bahawalpur, Dunyapur, Faisalabad,
Layyah, Multan and Karror have F/G ratio in the range of 1.58±0.18 to 1.71±0.11. Only one
beekeeper‟s honey B1 was more than 1.22±0.11. F/G ratio of adulterated (bee fed) honey
sample is 0.91. All other beekeeper‟s honey samples (B2-B5) were less than 1.14. F/G ratios
of beekeeper‟s honey samples were similar to adulterated honey sample. F/G ratios of honey
samples of local brands were also higher than 1.14. Most of Pakistani honeys analyzed in this
study have F/G ratios greater than 1.14 similar to Spanish, Argentinian and Venezuelian
honeys.
24.52
16.93
5.82
32.85
19.25
6.63
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
Fructose Glucose Maltose
D
G
Fru
cto
se, g
luco
se a
nd
mal
tose
g /
10
0 g
Honey bee Species
71
Table 4.1 Fructose glucose ratio of Pakistani honeys
Location F:G Location F:G
Abdulhakeem 1.58±0.18 IS1 1.49±0.01
Bahawalpur 1.66±0.07 IS2 1.63±0.01
Dunya Pur 1.66±0.02 Sulman‟s honey 0.00
Faisalabad 1.71±0.11 Marhaba 1.66±0.01
Layyah 1.67±0.07 China 1.73±0.01
Multan 1.66±0.03 Muqueet 1.72±0.01
Shorkot 1.68±0.02 Langanese 1.66±0.01
Karror 1.65±0.02 Sulman‟s honey 1.67±0.01
B1 1.22±0.01 Young‟s honey 1.68±0.01
B2 0.89±0.01 Life style honey 1.64±0.01
B3 0.93±0.01 Honey 1.66±0.01
B4 1.09±0.01 Honey of dwarf bee 1.47±0.24
B5 1.01±0.01 Honey of Giant bee 1.71±0.11
B6 1.06±0.01 Bee fed honey 0.91±0.03
I
72
II
III
Fig. 4.25a: HPLC chromatograms obtained for standards (maltose I, glucose II and
fructose III)
I
73
II
III
Fig. 4.25b: HPLC chromatograms obtained for beekeeper’s honey samples I, II and a
natural Sample III)
I
74
II
III
Fig. 4.25c: HPLC chromatograms obtained for some natural honey samples I, II, and
III)
Sugars profile of our natural honeys produced by Apis florea and Apis dorsata honey
bees collected from same locality are different. Fructose, glucose and maltose load of honey
produced by giant honey bee were higher as compared to dwarf honey bee as shown in Fig.
4.24. Honey samples produced by melponinae (stingless honeybee) contained surose, glucose
and fructose 3.72±0.49, 25.5±3.41 and 25.2±3.37 g/100 g of honey, respectively (Guerrini et
al., 2009). Results of honeys analyzed in our study are different from that reported for
melponinae (stingless honeybee).
Although fructose glucose ratio of honey is important parameter for honey
authenticity but detection of disaccharides (sucrose, turanose, kojibiose, maltulose and
isomaltos) and oligosaccharides is still required for origin and floral discrimination.
75
4.10 Mineral profile
4.10.1 Calcium (Ca)
Calcium levels of studied Pakistani honeys are given in Table 4.2. Calcium load in
natural Pakistani honeys were in the range of 80.49±0.83 to 149.11±24.00 µg/g with mean
value 100.59±22.53 µg/g. Honeys from the Shorkot have highest Ca content with mean value
of 149.11±4.78 µg/g while mean Ca load of honeys produced by Apis florea honey bee was
169.05±0.05 µg/g. In case of beekeeper‟s honey samples Ca load was much lower as
compared to all honey samples of natural localities and were found to be in the range of
13.5±0.45 to 65.38±2.18 µg/g. Ca levels of branded honey samples were variable. Ca
measured in Islamic shahed 1, Islamic shahed 2, China Honey, Langanese Honey, Young‟s
Honey, Life style and Honey were close to those found for beekeeper‟s honey samples. Ca
contents for Sulman‟s Honey, Muqeet Honey and Swat Honey were close to those for natural
honey sample while Marhaba Honey has higher Ca load as compared to all natural honey
samples. Our honey samples exhibited Calcium limits closer to the honey samples of Turky
(3.28±0.09-197±16.4 µg/g) (Silici et al., 2008), Brazil (1.25-150.20 µg/g) (dos Santos et al.,
2008) Poland (3.3±0.14-159.2±2.23 µg/g) (Madejczyk and Baralkiewicz, 2008), New
Zealand (7.21 to 94.30 µg/g) (Vanhanen et al., 2011), Syrian (43.30-118 µg/g) (Khuder et
al., 2010) and Galician honeys (a region of Spain) (91±31 to 185±41 µg/g) (Garcia et al.,
2006), Argentina (12.90±1.80 to 20.3±7.0 µg/g) (Baroni et al., 2009), Portugal (33.5±5.9 to
37.6±6.2 µg/g) (Almeida-Salvia et al., 2011) and Ireland (74.9 to 175 µg/g) (Downey et al.,
2005). Italian (137-409 µg/g) (Pisani et al., 2008) contained Ca more than our honeys.
Spanish honeys in two studies were found to contain Ca more than our honeys 42.59±4.40 to
341.0±31.87 µg/g (Fernandez-Torres et al., 2005) and 110-248 µg/g (Terrab et al., 2004;)
57.3±18-82.0±24.60 µg/g (Hernandez et al., 2005). In another study conducted by Gulfraz et
al. (2010) Ca limit in Pakistani honeys collected from hilly areas was in the range of
586.7±5.53 to 1035±61.49 µg/g which was much higher as compared to values obtained in
our study.
76
Table 4.2: Ca and Mg contents of studied Pakistani honeys
Types of Honeys Ca (Mean±SD; µg/g ) Mg (Mean±SD; µg/g)
Abdulhakeem 113.31±52.90 61.60±19.21
Bahawalpur 105.60±24.03 69.13±15.88
Dunya Pur 93.50±2.29 46.76±35.29
Faisalabad 84.23±5.90 63.67±20.90
Layyah 80.49±0.83 74.95±24.57
Multan 85.08±7.34 62.88±19.13
Shorkot 149.11±24.00 62.52±13.77
Karror 93.41±4.78 62.70±08.24
Dwarf 169.05±50.00 67.13±13.82
Beekeeper‟s Honeys 1 37.54±1.25 21.97±0.37
2 43.06±1.44 92.33±1.54
3 33.06±1.10 27.56±0.46
4 18.5±0.62 8.35±0.14
5 13.5±0.45 14.81±0.25
6 65.38±2.18 19.87±0.33
Islamic shahed 1 49.24±1.64 21.10±0.35
Islamic shahed 2 76.55±2.55 48.85±0.81
Sulman‟s Honey 90.78±3.03 21.79±0.36
Muqeet Honey 95.52±3.18 40.30±0.67
China Honey 62.73±2.09 14.46±0.24
Marhaba Honey 179.31±5.98 21.10±0.35
Langanese Honey 64.49±2.15 42.57±0.71
Swat Honey 123.83±4.13 25.98±0.43
Young‟s Honey 62.62±2.09 31.40±0.52
Life style 63.57±2.12 28.43±0.47
Honey 52.80±1.76 21.79±0.36
77
4.10.2 Magnesium (Mg)
Magnesium load of analyzed Pakistani honeys were given in Table 4.2 whereby
natural honeys were in the range of 46.76±35.29 to 74.95±24.57 µg/g with a mean value of
63.03±1.05 µg/g. Mg contents of honey samples produced by Apis florea honeybee were
67.13±13.82 µg/g whereas in adulterated honey samples those were 59.94±45.80 µg/g.
Beekeeper‟s honey samples contained Ca in the range of 8.35±0.14 to 92.33±1.54 µg/g. All
branded honey samples contained lower Mg load as compared to natural honey samples.
Honey samples of Argentina (9.40±5.9 to 17.30±19.40 µg/g) (Baroni et al., 2009),
Irland (18.9 to 53.3 µg/g) (Downey et al., 2005), Portugal (16.4±1.9 and 21.7±2.3 µg/g)
(Silva et al., 2009), Brazil (6.12 to 33.44 µg/g) (dos Santos et al., 2008), Poland (1.06±0.17-
21.3±0.54 µg/g) (Madejczyk and Baralkiewicz, 2008) and Turkey (21.9±2.0-67.5±4.80 µg/g)
(Silici et al., 2008) exhibited Mg less than our results whereas Mg contents in Galician (18-
308 µg/g) (Garcia et al., 2006), Spanish honeys in three studies (13.26±1.737 to
74.38±4.823 µg/g) (Fernandez-Torres et al., 2005), (28.40±29.70-49.60±33.0 µg/g)
(Hernandez et al., 2005), (37-139) µg/g (Terrab et al., 2004), New Zealand (7.52 to 86.30
µg/g) (Vanhanen et al., 2011) and Italian (22.2-159 µg/g) (Pisani et al., 2008) were close to
our honey samples.
4.10.3 Iron (Fe)
Iron profile of natural honeys collected from different locations was in the range of
7.71±3.05 to 27.72±3.22 µg/g as given in Table 4.3. Fe contents of honey produced by Apis
florea honey bee were higher as compared to honey produced by Apis dorsata honey bees of
same locality (Faisalabad, Pakistan). Fe contents of adulterated honey were only 6.74±1.20
µg/g and for of beekeeper‟s honey samples were in the range of 3.53±0.50 to 12.64±1.81µg/g
with a mean value of 7.61±3.41µg/g which was close to adulterated honey samples. In case
of branded honey samples amount of Fe was also very low except Swat and Young‟s honey
which have much higher Fe load. Our natural honey samples exhibited higher but our
commercial honey samples contained iron close to the honey samples of Argentina
(3.50±2.90 to 4.0±1.50 µg/g) (Baroni et al., 2009), Portugal (3.07±0.92 to 14.2±4.3 µg/g)
(Almeida-Salvia et al., 2011), New Zealand (0.67 to 3.39) (Vanhanen et al., 2011), Turkey
78
(1.12±0.10-12.0±1.10 µg/g) (Silici et al., 2008), Italy (0.97-13.7 µg/g) (Pisani et al., 2008)
and Galicia (2.0±0.8 µg/g) (Garcia et al., 2006). Our honey samples contained less iron as
compared to the Brazilian (1.50-89.08) (dos Santos et al., 2008), Egyptian (58±0.4 to
3690±79 µg/g) (Rashed and Soltan, 2004). Iron contents (128.7±2.92 to 224.4±2.49 µg/g) of
some Pakistani honeys determined by Gulfraz et al. (2010) were also greater than our
findings. Our results were close the Syrian (1.46 to 16.9 µg/g) (Khuder et al., 2010), Poland
(1.0±0.1-16.1±0.3 µg/g) (Madejczyk and Baralkiewicz, 2008), Spanish (1.52±0.30 to
7.64±11.63 µg/g) (Hernandez et al., 2005) and Irish (1.7 to 36.3 µg/g) (Downey et al., 2005)
honeys.
4.10.4 Zinc (Zn)
Zinc contents of studied honeys are given in Table 4.3. Zn contents in natural
Pakistani honeys, beekeeper‟s and branded honeys were in the range of 4.18±0.97 to
6.76±1.02, 0.64±0.005 to 2.12±0.006 and 0.12±0.00 to 1.41±0.03 µg/g, respectively. Mean
Zn contents of honeys produced by Apis florea and Apis dorsata honey bees of same locality
were 4.19±1.10 and 2.72±0.57, respectively. Our honey samples contained Zn contents close
to the honey samples of Poland (0.13±0.01 to 9.93±0.21) (Madejczyk and Baralkiewicz,
2008), Egypt (5.0±1.0 to 9.3±0.50) (Rashed and Soltan, 2004), Brazil (0.25 to 14.26) (dos
Santos et al., 2008) and Turkey (0.47±0.02 to 6.57±0.61 µg/g) (Silici et al., 2008). On the
other hand honey samples of Argentina (2.20±1.30 and 2.40±2.30 µg/g), (Baroni et al.,
2009), Italy (0.72 to 3.66 µg/g) (Pisani et al., 2008), Ireland (1.6 to 22.5 µg/g) (Downey et
al., 2005), Portugal (0.41±0.12 to 0.90±0.22 µg/g) (Silva et al., 2009), New Zealand (0.20 to
2.46 µg/g) (Vanhanen et al., 2011), Galician (2.2±1.1) (Garcia et al., 2006) and Syria (0.206
to 2.76) (Khuder et al., 2010) contained less Zn contents as compared to our honey samples.
Zinc contents in Pakistani honeys determined previously by Gulfraz et al. (2010) were much
higher (24.50±0.60 to 33.0±0.54 µg/g) as compared to our honey samples. Zinc found in
seven commercial honeys from seven countries Austria, Pakistan, Canada, Germany,
Australia, Saudi Arabia and USA was 0.2992, 0.3011, 0.1368, 0.4717, 0.2194, 0.110 and
0.1903, respectively were less than our findings (Bibi et al., 2008). Spanish honeys in one
study (1.332±0.006 to 7.825±1.167) (Fernandez-Torres et al., 2005) were comparable to our
79
natural honey while in another study (1.18±0.46 to 2.43±2.40 µg/g) Hernandez et al., 2005)
were less than our natural honeys but comparable to our commercial honey samples.
4.10.5 Manganese (Mn)
Manganese profile of studied Pakistani honeys is given in Table 4.3. Mn levels of
natural Pakistani honeys were 1.06±0.48 to 2.33±1.03. Mn contents in honeys produced by
Apis florae and Apis dorsata honey bees of the same locality (Faisalabad, Pakistan) were
1.74±0.84 and 1.06±0.48 µg/g, repectively while in case of adulterated honey sample it was
0.40±0.21 µg/g. Beekeeper‟s honey samples contained Mn in the range of 0.07±0.00 to
0.51±0.03 and branded honey samples as 0.12±0.01 to 1.41±0.07 µg/g. Overall Mn level in
commercial and adulterated honey samples were less as compared to natural honey samples.
Mn contents of our honey samples were closer to those of Syrian (0.153 to 1.69 µg/g)
(Khuder et al., 2010), Irish (0.9 to 10.2 µg/g) (Downey et al., 2005). and New Zealand (0.18
to 4.75 µg/g) (Vanhanen et al., 2011) honey samples whereas Argentinian (0.12±0.29 to
0.17±0.20 µg/g) (Baroni et al., 2009), Portugal (0.64±0.14 to 0.83±0.18 µg/g) (Almeida-
Silva et al., 2009) and Egyptian (0.50±1 to 1.70±0.25 µg/g) (Rashed and Soltan, 2004)
honeys have less Mn as compared to our natural honeys but close to our beekeeper‟s honey
samples. Mn contents of some Pakistani honeys determined by Gulfraz et al. (2010) were
slightly more than our honey samples 2.31±0.03 to 3.76±0.03 µg/g. Brazilian (0.90 to 52.98
µg/g) (dos Santos et al., 2008), Galician (0.5 to 20 µg/g) (Garcia et al., 2006), Rhododendron
honeys of Turkey (1.11±0.12 to 74.2±4.3 µg/g) (Silici et al., 2008), honeys of Poland were
0.17±0.1 to 7.73±0.20 µg/g (Madejczyk and Baralkiewicz, 2008). Italy honeys contain 0.13
to16.9 µg/g (Pisani et al., 2008) and Spain (0.20±0.06 to 9.47±0.35 µg/g) (Fernandez-Torres
et al., 2005) were more than our honey samples. Mn contents determined by Bibi et al.
(2008) in seven commercial honeys from seven countries Austria, Pakistan, Canada,
Germany, Australia, Saudi Arabia and USA were 0.081, 0.075, 0.070, 0.140, 0.341, 0.086
and 0.064 µg/g, respectively. All these samples contained less Mn as compare to our natural
honey samples but close to our commercial honey samples.
80
Table 4.3: Iron, manganese and zinc contents of studied Pakistani honeys
Types of Honeys Fe (Mean±SD;
µg/g)
Mn (Mean±SD;
µg/g)
Zn (Mean±SD;
µg/g)
Abdulhakeem 12.15±4.50 1.25±0.55 3.74±1.01
Bahawalpur 27.72±3.22 2.33±1.03 5.10±1.11
Dunya Pur 9.15±1.83 1.17±0.15 4.02±1.01
Faisalabad (Apis dorsata) 10.51±4.85 1.06±0.48 2.72±0.57
Layyah 9.39±0.90 1.29±0.11 2.77±0.05
Multan 7.71±3.05 1.40±0.39 4.28±1.08
Shorkot 27.12±1.37 2.07±0.20 6.76±1.02
Karror 8.06±1.49 1.17±0.11 4.18±0.97
Faisalabad (Apis florea) 21.97±9.96 1.74±0.84 4.19±1.10
Beekeeper‟s Honeys1 5.47±0.78 0.18±0.01 1.14±0.003
B2 5.89±0.84 0.07±0.00 0.64±0.005
B3 7.58±1.08 0.72±0.04 1.45±0.001
B4 10.56±1.51 0.51±0.03 0.68±0.009
B5 12.64±1.81 0.31±0.02 1.48±0.007
B6 3.53±0.50 0.12±0.01 2.12±0.006
Islamic Shahed 1 5.22±0.75 0.22±0.01 1.15±0.00
Islamic Shahed 2 7.72±1.10 0.18±0.01 1.25±0.00
Sulman‟s honey 3.28±0.47 0.26±0.01 2.18±0.01
Muqeet honey 2.31±0.33 0.12±0.01 1.13±0.00
China Honey 4.78±0.68 0.19±0.01 1.01±0.00
Marhaba Honey 3.56±0.51 1.41±0.07 1.19±0.03
Langanese Honey 3.17±0.45 0.25±0.01 1.78±0.01
Swat Honey 77.72±11.10 1.22±0.06 1.37±0.02
Young‟s honey 29.81±4.26 1.28±0.06 1.93±0.03
Life style 4.03±0.58 0.26±0.01 1.70±0.01
Honey 5.39±0.77 0.12±0.01 9.56±0.00
81
4.11 Bio indication of heavy metal toxicity of environment
World Health Organization (WHO) and Food and Agriculture Organization have
jointly recommended Provisional Tolerable Weekly Intake (PTWI) for toxic sustances which
can be safely ingested per week. PTWI were assigned for the toxic metals which can
accumulate in different body organs after digestion. WHO and FAO assigned PTWI to aware
the people about ingestion of foods containing these metals. PTWI values for Pb, Hg, Cd and
As are 25, 5, 7 and 15 µg/kg (Anonymous, 2000; Anonymous, 2005; Bilandzic et al., 2011).
Copper, cobalt and nickel analysis on honeys of north and south origins of Argentina
concluded that levels are below limits of detection in both origins (Baroni et al., 2009).
Limits recommended by Czech Bylaw for Cd, Hg, Pb, Zn and Cu are (0.5 µg/g), (0.5 µg/g),
(8.0 µg/g), (80.0 µg/g) and (80.0 µg/g), respectively (Celechovska and Vorlova, 2001).
4.11.1 Copper (Cu)
Copper level of all studied honeys is given in Table 4.4. Copper was found in almost
all analyzed samples. Recommended limit of Cu 80.0 µg/g (Celechovska and Vorlova,
2001). Copper concentration in Certified Reference Material is 5.64 µg/g (Silici et al., 2008).
Copper contents of honeys of Abdulhakeem, Dunya Pur, Faisalabad, Layyah and Shorkot
were less than recommended limit. Many honey samples of Bahawalpur, Multan and Karror
exhibited higher copper levels. Four samples of Bahawalpur, three samples of Multan and
two samples of Karror contained Cu more than the permissible limit. This may be due to
industrial interruption, herbicide, pesticides, fungicide and bactericide applications in
surrounding environment or copper equipment used in these localities. In case of commercial
honey samples, copper levels were less than recommended levels in all beekeeper‟s honey
samples as well as in branded honey samples.
High copper levels may be due to equipment used by beekeepers during honey
processing (dos Santos et al., 2008). Provisional tolerable daily intake (PTDI) value of Cu for
a 60 kg adult person is 3 mg (Anonymous, 2000). Presence of copper in food items may be
due to crop mineralization, contamination of environment, herbicide, pesticides, fungicide
and bactericide applications in surrounding environment (Provenzano et al., 2010: Bilandzic
et al., 2011). Cu contents of honeys are 1 to 2.3 µg/g (Downey et al., 2005). Silva et al.
82
(2009) analyzed that Cu values of Portugal honeys were different for different locations. Cu
contents in wild and orchard Portugal honeys were 0.405±0.014 and 0.74±0.21 µg/g (Silva et
al., 2009). Cu in New Zealand honeys was in the range of 0.09 to 0.70 with a mean value of
0.25 µg/g (Vanhanen et al., 2011). Cu contents of five regions of Croatia were determined
while high cocentrations were detected only in some areas with a mean value (1074 µg/kg).
Highest copper contents were found in Centre region with a mean value of 3232 µg/kg.
Southwest and Centre regions also faced high Cu levels. Despite the fact that low Cu levels
were observed in the Northeast regions (Bilandzic et al., 2011). Cu contents of some
Pakistani honeys determined by Gulfraz et al. (2010) were in the range of 12.42±0.25 to
18.33±0.29 µg/g within permissible level. Copper contents found in seven commercial
honeys from seven countries Austria, Pakistan, Canada, Germany, Australia, Saudi Arabia
and USA were 0.498, 0.494, 0.026, 0.164, 0.033, 0.023 and 0.032, respectively (Bibi et al.,
2008). Italian, Galician, Syrian, Poland and Czech Republic honeys contained 172 to 59000
µg/kg, 0.08 to 2.0 µg/g, 0.616 to 2.21µg/g to 10.5±0.8-29.5±2.1 µg/kg, 0.26±0.05 to
1.82±0.09 µg/g and 0.06 to 1.55 µg/kg Cu (Celechovska and Vorlova, 2001; Garcia et al.,
2006; Madejczyk and Baralkiewicz, 2008; Pisani et al., 2008; Silici et al., 2008; Khuder et
al., 2010). Cu values in Spanish honeys analyzed in two studies were 0.53 to 2.12 and
0.20±0.06 to 0.44±0.28 µg/g (Fernandez-Torres et al., 2005; Hernandez et al., 2005).
Egyptian honeys contained 1.0±0.0 to 1.75±0.35 µg of copper per g of honey (Rashed and
Soltan, 2004).
4.11.2 Lead (Pb)
Lead load in honey samples of the Karror and Shorkot was less than that of
permissible limits. Recommended in limit is 0.47 µg/g (Silici et al., 2008) Lead contents of
analyzed Pakistani honeys are given in the table 4.3. One honey sample from Bahawalpur
showed 2.05±0.10 µg/g, three honey samples from Faisalabad contained 0.68±0.03,
1.93±0.09 and 2.39±0.11 µg/g Pb, two honey samples of Multan contained 0.68±0.03 and
3.75±0.18 µg/g of Pb. All other analyzed honey samples from Faisalabad, Multan,
Bahawalpur, Layyah and Dunya pur contained Pb less than recommended level. In case of
commercial honey samples one beekeepe‟s honey contained 1.59±0.08 µg/g, Isalamic shahed
contained 1.93±0.09 µg/g and Young´s honey contained 0.57±0.03 µg/g of lead while all
83
other commercial honey samples also contained under the recommended limits. Pb contents
in New Zealand honeys were in the range of 0.01 to 0.04 with a mean value of 0.017µg/g
(Vanhanen et al., 2011). Lead contents in Croatian honeys were detectedin five locations and
found in all regions. Mean values were 65.2 µg/kg. Lead contents in East and Centre regions
of Croatia were 23.4 and 131 µg/kg. High lead contents may be due to presence of railways
and highways near the beehives. Honey samples from Poland, Slovenia and Romania were
25 to 70, 1.86 to 4.3 and 0.07 µg/kg, respectively (Przybylowski and Wilczynscka, 2001)
while Turkish honeys contained 17.6 to 32.1 and 8.4 to 106 µg/kg determined in two
different studies by Tuzen and Soylak (2005) and Tuzen et al. (2007), respectively. A study
conducted on Italian honeys in 1982 was very useful to highlight Pb toxicity in an area of
Italy. Mean value of Pb was 2370 µg/kg detected in Croatian honey samples (Bilandzic et al.,
2011). Lead level was determined in twenty Rhododendron honeys of Turkey were in the
range of 1.54±0.11 to 55.3±4.73 µg/kg (Silici et al., 2008). Lead found in seven commercial
honeys from seven countries were 0.28, 0.17, 0.04, 0.12, 0.02, 1.81 and 0.24 for honeys
samples from Austria, Pakistan, Canada, Germany, Australia, Saudi Arabia and USA,
respectively (Bibi et al., 2008). Italian, Galician and Czech Republic honeys contained 28.2
to 304 µg/kg, 2.0-49 ng/g and 0.02-1.0 µg/g lead (Celechovska and Vorlova, 2001; Garcia et
al., 2006; Pisani et al., 2008). Egyptian honeys contained 4.2±0.35-6.3±1 µg of lead per g of
honey while syrup fed honeys contained 9.3 µg/g of honey (Rashed and Soltan, 2004).
4.11.3 Cobalt (Co)
Permissible concentration of cobalt in is 0.30 µg/g (Silici et al., 2008). Cobalt limits
of studied honey samples were given in Table 4.4. Two samples of Abdulhakeem contained
0.32±0.019 and 0.38±0.023 µg/g, three samples of Bahawalpur contained 0.38±0.023,
0.38±0.023 and 0.32±0.019 µg/g, one sample of Faisalabad contained 0.51±0.030 µg/g, three
samples of Multan contained 0.42±0.025, 0.51±0.030 and 0.35±0.021 µg/g of cobalt. All
other honey samples also contained cobalt in significant amount but less than recommended
84
Table 4.4: Copper, lead and cobalt contents of studied Pakistani honeys
Continued……..
Locality of
Honey Sample
No. of
Samples
Cu (Mean±SD;
µg/g)
Pb(Mean± SD;
µg/g)
Co (Mean±SD;
µg/g)
Abdulhakeem 1 2.46±0.13 0.00±0.00 0.06±0.004
2 2.14±0.11 0.00±0.00 0.13±0.008
3 0.93±0.05 0.57±0.03 0.32±0.019
4 1.45±0.08 0.46±0.02 0.19±0.011
Bahawalpur 1 7.06±0.37 0.00±0.00 0.38±0.023
2 6.65±0.35 0.00±0.00 0.38±0.023
3 4.56±0.24 0.00±0.00 0.32±0.019
4 5.97±0.31 0.00±0.00 0.19±0.011
5 9.27±0.49 2.05±0.10 0.29±0.017
6 0.81±0.04 0.00±0.00 0.22±0.013
7 2.66±0.14 0.00±0.00 0.16±0.009
Dunya Pur 1 2.82±0.15 0.00±0.00 0.16±0.009
2 0.56±0.03 0.57±0.03 0.22±0.013
3 0.89±0.05 0.00±0.00 0.22±0.013
4 5.12±0.27 0.00±0.00 0.19±0.011
Faisalabad 1 2.06±0.11 1.93±0.09 0.06±0.004
2 1.77±0.09 2.39±0.11 0.13±0.008
3 1.77±0.09 0.34±0.02 0.19±0.011
4 1.09±0.06 0.34±0.02 0.29±0.017
5 2.86±0.15 0.00±0.00 0.06±0.004
6 1.09±0.06 0.00±0.00 0.29±0.017
7 3.71±0.20 0.68±0.03 0.03±0.002
8 1.98±0.10 0.00±0.00 0.51±0.030
9 1.21±0.06 0.00±0.00 0.03±0.002
85
Continued………….
Locality of
Honey Sample
No. of
Samples
Cu (Mean±SD;
µg/g)
Pb (Mean±SD;
µg/g)
Co (Mean±SD;
µg/g)
10 0.93±0.05 0.00±0.00 0.16±0.009
11 3.15±0.17 0.46±0.02 0.19±0.011
12 1.73±0.09 0.00±0.00 0.16±0.009
Layyah 1 1.21±0.06 0.00±0.00 0.03±0.002
2 2.14±0.11 0.00±0.00 0.22±0.013
Multan 1 4.15±0.22 0.00±0.00 0.22±0.013
2 1.53±0.08 0.11±0.01 0.29±0.017
3 13.19±0.69 0.11±0.01 0.19±0.011
4 6.05±0.32 0.00±0.00 0.42±0.025
5 5.12±0.27 0.00±0.00 0.16±0.009
6 7.14±0.38 0.68±0.03 0.51±0.030
7 1.01±0.05 0.00±0.00 0.03±0.002
8 0.89±0.05 3.75±0.18 0.35±0.021
9 1.09±0.06 0.00±0.00 0.03±0.002
Shorkot 1 1.77±0.09 0.00±0.00 0.38±0.023
2 1.94±0.10 0.00±0.00 0.13±0.008
3 1.09±0.06 0.00±0.00 0.16±0.009
4 2.46±0.13 0.00±0.00 0.00±0.000
Karror 1 5.12±0.27 0.11±0.01 0.32±0.019
2 6.45±0.34 0.00±0.00 0.16±0.009
3 6.05±0.32 0.00±0.00 0.22±0.013
Beekeepers 1 1.29±0.07 0.00±0.00 0.00±0.000
2 0.48±0.03 0.00±0.00 0.48±0.028
3 0.65±0.03 1.59±0.08 0.29±0.017
4 0.60±0.03 0.34±0.02 0.22±0.013
5 1.05±0.06 0.00±0.00 0.19±0.011
6 1.77±0.09 0.00±0.00 0.19±0.011
86
level. Review of cobalt analysis in all honey samples indicated the presence of cobalt risk in
the respective areas. In case of commercial honey samples only one beekeeper‟s honey,
Marhaba honey and Honey (named as honey) contained 0.48±0.028, 0.32±0.019 and
0.35±0.021 µg/g cobalt, respectively. All other commercial honey samples contained less
amount of cobalt. Co level determined in twenty Rhododendron honeys of Turkey was in the
range of 1.25±0.10 to 28.5±2.10 µg/kg (Silici et al., 2008). Italian honeys contained 1.56 to
56.6 µg/kg cobalt (Pisani et al., 2008). Egyptian honeys contained 1.75±0.35 to 2.5±0.70 µg
of pottasium per g of honey.
4.11.4 Cadmium (Cd)
Cd concentrations in studied Pakistani honeys are given in Table 4.5. Recommended
concentration of cadmium in is 0.013µg/g. Cadmium load in two honey samples of
Abdulhakeem were 0.13±.004 and 0.07±0.004 µg/g, four honey samples of Bahawalpur
contained 0.02±0.001, 0.02±0.001, 0.27±.001 and 0.02±0.001 µg/g, three honey samples of
Dunya pur contained 0.02±0.001, 0.05±0.003 and 0.04±0.002 µg/g, seven honey samples of
Faisalabad contained 0.02±0.001, 0.04±0.002, 0.03±0.002, 0.02±0.001, 0.03±0.002,
0.03±0.002 and 0.13±.005 µg/g, one honey sample contained 0.02±0.001, 0.04±0.002 µg/g,
Locality of
Honey Sample
No. of
Samples
Cu (Mean±SD;
µg/g)
Pb (Mean±SD;
µg/g)
Co (Mean±SD;
µg/g)
Islamic Shahed 1 1.09±0.06 0.68±0.03 0.03±0.002
2 2.46±0.13 1.93±0.09 0.16±0.009
Salman´s Honey 1 0.65±0.03 0.00±0.00 0.00±0.000
Marhaba Honey 1 0.16±0.01 0.00±0.00 0.32±0.019
China Honey 1 1.05±0.06 0.00±0.00 0.22±0.013
Muqeet Honey 1 0.77±0.04 0.00±0.00 0.22±0.013
Langanese
Honey
1 1.01±0.05 0.000.00 0.19±0.011
Swat Honey 1 0.81±0.04 0.00±0.00 0.06±0.004
Young´s Honey 1 0.74±0.11 0.57±0.03 0.13±0.008
Life Style 1 0.65±0.03 0.00±0.00 0.16±0.009
Honey 1 1.09±0.06 0.34±0.02 0.35±0.021
87
Table 4.5: Cadmium, cromium and manganese contents of studied Pakistani honeys
Continued.......
Types of
Samples
No. of
Samples
Cd (Mean±SD;
µg/g)
Cr (Mean±SD;
µg/g)
Ni (Mean±SD;
µg/g )
Abdulhakeem 1 0.00±0.000 1.09±0.05 0.45±0.003
2 0.01±0.001 0.70±0.03 0.875±0.01
3 0.13±.004 0.00±0.00 0.25±0.002
4 0.07±0.004 0.00±0.00 0.4±0.003
Bahawalpur 1 0.02±0.001 1.17±0.05 0.55±0.004
2 0.01±0.001 1.41±0.06 0.55±0.004
3 0.02±0.001 0.00±0.00 0.13±0.001
4 0.27±.001 0.00±0.00 0.40±0.003
5 0.02±0.001 1.09±0.05 2.53±0.017
6 0.00±0.000 0.00±0.00 0.65±0.004
7 0.01±0.000 0.00±0.00 0.38±0.002
Dunya Pur 1 0.02±0.001 1.25±0.05 1.23±0.008
2 0.05±0.003 1.25±0.05 0.37±0.002
3 0.04±0.002 1.17±0.05 0.15±0.001
4 0.01±0.000 0.08±0.00 0.45±0.003
Faisalabad 1 0.02±0.001 1.09±0.05 0.65±0.004
2 0.04±0.002 1.25±0.05 0.68±0.004
3 0.03±0.002 1.09±0.05 0.53±0.003
4 0.02±0.001 0.86±0.04 0.40±0.003
5 0.00±0.000 1.17±0.05 4.93±0.033
6 0.01±0.001 0.00±0.00 0.55±0.004
7 0.00±0.000 1.09±0.05 2.03±0.013
8 0.01±0.001 1.09±0.05 2.05±0.014
88
Continued.......
Types of Sample No. of
Sample
Cd (Mean±SD;
µg/g)
Cr (Mean±SD;
µg/g)
Ni (Mean±SD;
µg/g)
9 0.00±0.000 0.08±0.00 0.25±0.002
10 0.03±0.002 1.17±0.05 0.58±0.004
11 0.13±.005 1.17±0.05 0.53±0.003
12 0.03±0.002 0.00±0.00 0.85±0.006
Layyah 1 0.01±0.001 0.00±0.00 0.55±0.004
2 0.02±0.001 0.00±0.00 0.18±0.001
Multan 1 0.01±0.000 0.78±0.03 0.85±0.006
2 0.04±0.002 1.33±0.06 0.025±0.002
3 0.07±0.004 1.56±0.07 0.58±0.004
4 0.12±.005 0.94±0.04 0.83±0.005
5 0.27±.006 0.00±0.00 0.33±0.002
5 0.27±0.006 0.00±0.00 0.33±0.002
6 0.07±0.004 0.00±0.00 0.38±0.003
7 0.03±0.002 0.08±0.00 0.00±0.00
8 0.11±0.000 1.41±0.06 0.30±.001
9 0.01±0.002 0.00±0.00 0.98±.003
Shorkot 1 0.03±0.002 0.55±0.02 1.10±0.007
2 0.04±0.002 1.09±0.05 0.65±0.004
3 0.01±0.000 1.17±0.05 0.80±0.005
4 0.01±0.000 1.17±0.05 0.65±0.004
Karror 1 0.01±0.000 1.25±0.05 0.6±0.004
2 0.05±0.003 0.00±0.00 0.10±0.001
3 0.02±0.001 0.00±0.00 0.40±0.003
Beekeepers 1 0.00±0.000 0.00±0.00 0.18±0.00
2 0.02±0.001 0.00±0.00 0.00±0.00
3 0.13±.007 0.00±0.00 0.30±0.002
89
seven honey samples of Multan contained 0.07±0.004, 0.12±.005, 0.27±.006, 0.27±0.006,
0.07±0.004, 0.03±0.002 and 0.11±0.000 µg/g two honey samples of Shorkot contained
0.03±0.002 and 0.04±0.002 µg/g, two honey samples of Karror contained 0.05±0.003 and
0.02±0.001µg/g of Cd. In case of commercial honeys many honeys contained more than
0.013µg/g. Presence of high levels of Cd in many Pakistani honeys indicated the presence of
Cd toxicity in those honeys. Cd contents in the honeys from five regions of Croatia were in
the range of 1.00-24.0 µg/kg. Cd contents in New Zealand honeys were in the range of 0.01
to 0.45 with a mean value of 0.149 µg/g (Vanhanen et al., 2011). Southwest region of New
Zealand contained highest Cd level with a mean value of 2.11 µg/kg. Cd levels in honeys
from Romania, Macedonia and Italy were 0.015, 3.63 and 4.25 µg/kg, respectively (Bratu
and Bergescu, 2005; Stankovska et al., 2007; Pisani et al., 2008) while four studies on
honeys of different geographical origins of Turkey had found that Cd contents were
0.32,10.9-21.2, 1.1-17.90 and 0.38-2.03 µg/kg (Erbilir and Erdogrul, 2005; Tuzen and
Soylak, 2005; Tuzen et al., 2007; Silici et al., 2008). Cadmium contents were determined in
Types of Sample No. Of
Sample
Cd(Mean±SD)
µg/g
Cr(Mean±SD)
µg/ g
Ni(Mean±SD)
µg/g
4 0.01±0.001 0.00±0.00 0.73±0.005
5 0.03±0.002 0.00±0.00 0.10±0.001
6 0.02±0.001 0.00±0.00 0.00±0.00
Islamic Shahed 1 0.04±0.002 0.00±0.00 0.18±0.00
2 0.07±0.004 0.00±0.00 0.00±0.00
Salman´ Honey 1 0.04±0.002 0.00±0.00 0.30±0.002
Marhaba Honey 1 0.05±0.003 0.00±0.00 0.73±0.005
China Honey 1 0.00.0001 0.00±0.00 0.10±0.001
Muqeet Honey 1 0.04±0.002 0.00±0.00 0.00±0.00
Langanese Honey 1 0.04±0.002 0.00±0.00 0.45±0.003
Swat Honey 1 0.04±0.002 0.00±0.00 0.45±0.003
Young´s honey 1 0.03±0.002 0.00±0.00 0.18±0.001
Life Style 1 0.04±0.002 0.00±0.00 0.28±0.002
Honey 1 0.07±0.004 0.00±0.00 0.17±0.001
90
twenty Rhododendron honeys of Turkey were in the range of 0.28±0.02 to 2.37±0.20 µg/kg
(Silici et al., 2008). Italian honeys contained 1.00-15.30 µg/kg cadmium (Pisani et al., 2008).
Cd in Galician honey was in the range of 0.8-6.2 ng/g (Garcia et al., 2006). Egyptian honeys
contained 0.01±0.10-0.50±0.00 µg of cadmium per g of honey. Cd contents in Czech
Republic honeys were 0.5-77.4 µg/kg (Celechovska and Vorlova, 2001).
Cadmium levels in Pakistani honeys were higher than honeys of Croatia, Czech
Republic New Zealand, Romania, Macedonia, Turkey, Galicia and Italy. Pakistani honeys
contained Cd levels close to those found in Egyptian honeys.
4.11.5 Chromium (Cr)
Permissible concentration of Cr in is 0.30 µg/g (Silici et al., 2008). Chromium levels
of studied Pakistani honeys are given in Table 4.5. Cr level in two honeys of Abdulhakeem,
three honeys of Bahawalpur, three honey samples of Dunya Pur, nine honey samples of
Faisalabad, six honey samples of Multan, all samples of Shorkot and one honey sample of
Karor were more than 0.30 µg/g. Therefore, these areas have risk of Cr toxicity. In case of
commercial honey samples of beekeeper and branded honey samples have zero Cr
concentration.
Cr contents in New Zealand honeys were in the range of 0.12 to 0.55 with a mean
value of 0.37 µg/g and detected in three sources out of eleven anlalysed sources (Vanhanen
et al., 2011). Cr contents in Syrian honeys were <0.018 to 0.054 µg/g (Khuder et al., 2010).
Cr contents of honey samples from Poland were 0.0051±0.0002-0.0930±0.0009 µg/g
(Madejczyk and Baralkiewicz, 2008). Cr in Galician honey was in the range of 5.6-41ng/g
(Garcia et al., 2006). Cr contents of many natural Pakistani honeys of different localities
were close to those for New Zealand honeys.
4.11.6 Nickle (Ni)
Nickle permissible concentration is 0.91µg/g (Silici et al., 2008). Nickle
measurements are given in Table 4.5. One honey sample from Bahawalpur, one honey
sample from Dunyapur, three honey samples from Faisalabad and one honey sample from
Multan contained more than 0.91µg/g of Ni. Commercial honey samples of different brands
91
and beekeepers were also having low levels of Ni therefore these localities have no risk of Ni
toxicity. But three honey samples from Faisalabad showed 4.93±0.033, 2.03±0.013 and
2.05±0.014 µg/g of Ni which is much higher than recommended limit. Consequently some
locations of Faisalabad are at high risk of Ni toxicity. Ni contents of honey samples from
Poland, Galicia and Italy were 0.023±0.002 to 1.326±0.011µg/g, 12 to 172 ng/g and 77 to
2760 µg/kg (Garcia et al., 2006; Madejczyk and Baralkiewicz, 2008; Pisani et al., 2008).
Egyptian honeys contained 1.25±0.35 to 1.7±0.25 µg of Ni per g of honey. At the same time
as syrup fed honeys of Egypt had 3.0 µg/g of honey (Rashed and Soltan, 2004). Most
Pakistani honeys analyzed in current study contained low levels of Ni and similar to results
found for Egyptian, Poland, Galician and Italian honeys.
Although some honey samples from some regions contained heavy metals more than
permissible limits but this study is not enough as the only scale for assessment of heavy
metals environmental contamination. A detailed study on large number of honey samples as
well as bees wax, soil and water samples from each region is required for comprehensive
evaluation of heavy metal toxicity.
4.12 Isotopic ratio analysis of C12
/C13
for detection of adulteration
Ratio of C12
/C13
analysis of analyzed honeys is given in the table 4.6. δ13
CPDB values
of natural honey collected from different locations were in the range of -26.07±0.16 to -
27.96±0.15%. δ 13
CPDB values of honey samples collected from beekeepers were in the range
of -11.73±0.14 to -17.77±0.15%. δ13
CPDB value for honey produced by artificial feeding of
bees was -16 5±0.15%. δ13
CPDB for honey of different brands available in markets were in the
range of -16.85±0.15 to -26.05±0.15%. Black locust and lime honeys of Dinaric,
Mediterranean and Pannonia regions exhibited δ13
CPDB values -24.2,-24.8 and -24.8 ‰ & -
25.6, -25.4 and -26.2 ‰, respectively (Kropf et al., 2010). Flowering plants (C3) C13
/C12
ratio in the range of -32 to -21% while in corn and cane plants (C4) C13
/C12
ratio in the range
of -19 to -12%. Adulteration of corn and cane sugars in natural honeys disturbs original
C13
/C12
of natural sugars originated from flowering plant (Padovan et al., 2003; Kropf et al.,
2010; Schellenberg et al., 2010). Carbon isotopic ratio of natural Pakistani honeys were in
the range of recommended values for flowering plants while δ 13
CPDB values of all
92
Table 4.6: Isotopic ratio analysis of C12
/C13
in analyzed Pakistani honeys
Location of Sample δ13
CPDB Brand Name of Sample δ13
CPDB
Abdulhakeem -26.96±0.15 Islamic Shahed Centre -16.85±0.15
Bahawalpur -27.36±0.14 Salman´s Honey -21.95±0.14
Dunya Pur -27.14±0.15 Marhaba Honey -23.60±0.14
Faisalabad -26.07±0.16 China Honey -23.54±0.16
Layyah -27.76±0.11 Muqeet Honey -23.34±0.15
Multan -26.71±0.13 Langanese Honey -26.05±0.15
Shorkot -26.50±0.12 Swat Honey -23.11±0.15
Karror -27.25±0.15 Young´s Honey -24.10±0.14
Beekeeper‟s Honey -17.77±0.15 Life Style -23.07±0.15
Beekeeper‟s Honey -11.96±0.16 Cooperative -25.09±0.15
Beekeeper‟s Honey -11.73±0.14 Beekeeper‟s Honey -15.77±0.13
Bee Fed Honey -16.85±0.15 Beekeeper‟s Honey -13.66±0.11
beekeeper‟s honeys indicated the adulteration by beefed with cane and beet sugars. While δ
13CPDB values of most of branded honeys indicate the adulteration by mixing the cane and
beet inverts in pure honeys. Langanese honey exihibited δ 13
CPDB equal to natural honeys.
Honey samples exhibited the δ 13
CPDB in the range of flowering plants are highlighted as pink
while those exihibited the δ 13
CPDB close to artificial honeys are highlighted as blue in the
table 4.6.
4.13 Statistical Analysis
4.13.1 Principal component analysis
Biplot of the first two principal components showed that natural honeys of Faisalabad (Fsd),
Multan (Mul) and Abdulhakeem (Ab) were associated with high Ca level while honey
samples of Shorkot (Sh) and Bahawalpur (Ba) were associated with Mn contents, electrical
conductivity (EC) and d13CPDB. Natural honeys of Layyah (Lay) are rich in Mg contents
while Karror (Ka) and Dunya pur (Du) honey samples were associated with high values of
Zn and glucose. Many beekeepers‟ honey samples and some commercial honey samples Is1,
93
Is2, Ma, Co, Li contained low amount of Ca, Mn, Fe and exhibited low EC values.
Adulterated (A), La and two beekeeper‟s honey samples are associated with high pH values.
Silvano et al. (2014) proposed that the variable located further away from origin point have
more contribution in variability. Glucose, Fructose, Moisture, F/G, Mn, Mg and refractive
index are the major variables in our study as shown in the Fig. 26.
Fig. 4.26: Biplot of principal component analysis of natural, branded and beekeeper’s
honey samples
Eigen values and variance as shown in the table proposed that first six principal
components can satisfactorily explain the most of the physicochemical properties of honey
samples. First six components explain 85% of the total variance. PC1 explains 30.4% of total
variance and major variables responsible for the variation are Mn, fructose, refractive index
94
(Rf) and fructose/glucose. PC2 explains 18.4% of total variance and major variables are
fructose, Mg and glucose. PC3 suggested that pH and EC while PC4 selects the d13CPDB as
major variable. According to PC5 Mg and d13CPDB while according to PC6 pH is the major
variable.
Table 4.7: Results of the principal component analysis on the physicochemical
properties
PC1 PC2 PC3 PC4 PC5 PC6
Eigen value 4.5574 2.6716 2.1083 1.2085 1.1647 1.0183
Variance (%) 0.304 0.178 0.141 0.081 0.078 0.068
Cumulative
Variance
0.304 0.482 0.622 0.703 0.781 0.849
Zn µg/g 0.284 -0.019 0.195 0.289 -0.028 0.378
Ca µg/g 0.304 0.184 -0.341 0.077 -0.112 0.128
Mg µg/g 0.160 0.381 0.159 -0.280 0.343 0.156
Mn µg/g 0.340 0.303 0.061 0.235 -0.135 -0.055
Fructose 0.325 -0.383 -0.127 -0.111 -0.078 -0.060
Glucose 0.158 -0.497 0.101 -0.113 -0.178 -0.180
Maltose 0.007 -0.043 0.301 0.101 -0.743 0.251
Fructose/glucose 0.382 -0.081 -0.302 -0.018 -0.010 0.049
Rf index -0.376 0.099 -0.021 0.142 -0.201 -0.175
pH -0.101 0.249 -0.415 -0.127 -0.223 0.502
EC mS/cm 0.150 0.243 0.484 -0.263 -0.136 -0.156
Moisture % 0.374 -0.184 -0.011 0.021 0.155 0.016
Ash 0.289 0.302 0.178 -0.193 -0.087 -0.222
d13CPDB 0.028 0.132 -0.371 -0.487 -0.342 -0.360
4.13.2 Cluster Analysis
Fig. 4.27 shows the arrangement of honey samples into five clusters. Natural honey
samples of Abdulhakeem (Ab), Bahawalpur (Ba), Multan (M), Shorkot (Sh) and Karror (Ka)
95
were classified in the first cluster including 20% of total samples. The average values of
samples in this cluster for Zn, Mn, EC, d13CPDB and moisture% were higher than the mean
of all other variables. Natural honey samples of Dunya pur (Du), Faisalabad (Fsd), Layyah
(Lay) and commercial honey samples IS2, Ma, and Li were classified in cluster 2. Honey
samples belongs to cluster two were characterized by high fructose contents and d13CPDB.
Adulterated, one beekeeper‟s honey and La honey were classified in third cluster these honey
samples had high Mg contents while lower fructose, glucose, maltose contents and less
d13CPDB values. All beekeeper‟s honey samples except one sample and Is1, Ch, Yo and Co
honey samples were included in the fourth cluster all these honey samples were rich in
glucose and maltose while poor in Ca and Mg levels. Cluster five includes two commercial
honey samples Sw and Mu. Honey samples of fifth cluster were exhibited high pH, F/G, Fe
and Ca contents.
Fig. 4.27: Dandrogram of cluster analysis
The most similar clusters were cluster three and four and the largest intercluster distance was
between cluster four and five. This result showed that Fructose, glucose, maltose, d13CPDB,
Mg, Ca, F/G are the most important variables in differentiating the honey samples.
Y o C o C h B 6 B 5 B 4 B 3 I s 1 B 1 B 2 L a A L ay
F s d L i I s 2 M a D u
S w M u S h K a M B a A b
-134.81
-56.54
21.73
100.00
Observations
Dendrogram Ward Linkage, Euclidean Distance
S
imila
rity
96
4.2 Bioactive components of honeys
Bioactive properties of honey mainly based on botanical composition of environment
(Wei and Zhirong, 2003; Kucuk et al., 2007; Isla et al., 2011) except some enzymes which
were added by honey bee during honey maturation and nectar collection. Honey enzymes
also showed certain bioactive properties (Michael et al., 2002; Huidobro et al., 2005; Al et
al., 2009). Bioactive contents of natural and commercial honeys were point of difference
among natural and commercial honeys. Because bioactive contents like flavonoids, phenolics
and carotene were in lesser amount in branded and beekeeper‟s samples than natural samples.
All types of honeys have shown good bioactive properties like antibacterial, antioxidant and
antitumor properties even in the case of beekeeper‟s honey samples this may be due to
presence of honey bee enzymes.
4.2.1 Total phenolic contents (TPC)
Total phenolic contents determined by Folin-Ciocalteu method was expressed as mg gallic
acid equivalent (GAE) per 100 g of honey. Natural Pakistani honeys shown in Fig. 4.28 from
different localities were found to be in the range of 55.41 – 165.61 mg GAE/ 100 g honey.
Honey samples collected from the Bahawalpur has maximum phenolic contents. On the other
hand minimum phenolic contents were found in honeys from Faisalabad. Phenolic contents
of honeys from Layyah and Karror were more than100 mg/100 g of honey. Isla et al. (2011)
analyzed phenolic contents of thirteen honeys form different Provinces of Argentina and
found that phenolic contents were in the range of 187.30- 1073.21µg GAE/ g. They found
that phenolic contents depend on floral origin of honeys. Multifloral honeys contained higher
phenolic contents as compared to monofloral honeys. Phenolic contents for 20 Canadian
honeys were in the range of 49.78±13.61-513.25±8.03 µg GAE/g of honey. Phenolic
contents of their honeys depend on the color of honey which in turn depends on the floral
origin of honeys. Saxena et al. (2010) also found that phenolic contents of seven commercial
honeys depended on floral origin. TPC values were in the range of 47±0.2-98±1.2 mg GAE
/100 g. Lachman et al. (2010) analyzed the TPC of forty honeys from different locations of
Czech Republic. They found that total phenolic contents of honey were depending on locality
and botanical origin of honey.
97
56.69
165.61
64.07 55.41
127.65
85.82 71.29
110.55
0
50
100
150
200
250
TPC
(m
g/1
00
gm
)
Sampling Areas
Fig. 4.28: Total phenolic contents of natural Pakistani honeys collected from some
selected locations
Overall TPC of their honeys was in the range of 82.5-242.5 mg GAE/kg of honey.
TPC of Turkish honeys from different locations were significantly different. Giresun honeys
contained highest phenolic contents as compared to honeys from other geographical regions.
TPC values for fifty honeys were in range of 0.24-141.83 mg/100 g (Silici et al., 2010).
Ferreira et al. (2009) analyzed three Portugal honeys and found that TPC values were in the
range of 226.16±0.22- 727.77±0.23 mg GAE/kg while in another study Portugal honeys were
found to contain 114 - 300 mg/kg. Al et al. (2009) analyzed twenty four Romanian honeys
from different locations and found to contained 2 to 125.00 mg GAE/ 100g. They concluded
that TPC was dependent on geographical origin. Venezuelan honeys contained 38.15-182.10
mg GAE/100 g (Vit et al., 2009). Slovenian honeys contained 27.7-285.7 GAE mg/kg
(Bertoncelj et al., 2007). Italian honeys from different localities were analyzed by Buratti et
al. (2007). Those natural honeys revealed 17.1±0.7 to 60±1 mg/100 g. Blasa et al. (2007)
analyzed Italian honeys collected from beekeepers and found TPC in the range of 3-17.50
mg/100 g. Overview of total phenolic contents of different countries has concluded that
Pakistani natural honeys were richest in phenolics. TPC values of natural honeys were much
higher than international honeys. Our results are comparable to Venezuelean, Indian and
Portugal honeys.
98
In case of commercial honeys TPC values of almost all honeys were very much
different as compared to natural honeys as shown in Fig. 4.29. Mean TPC of natural honeys
was 89.81 mg GAE/100 g. Bar A indicated the mean TPC of adulterated honeys. Most of
beekeeper‟s honeys and many branded honeys showed TPC measurements close to
adulterated honeys. Only one study conducted by Ferreira et al. (2009) determined in pure
and extracted form. Amount of total phenolics in Portugal‟s honeys analyzed in pure form
were 226.16±0.22 mg GAE/100 g, 406.23±17.22 GAE/100 g and 727.77±0.23 GAE/100 g
for light, amber and dark honeys respectively while that study also found out that total
phenolic contents determined after isolation with amberlite column were lower than
determined in pure honey. Therefore, TPC results are more reliable if determined after
extraction of phenolics with the help of amberlite columns. Overall Pakistani honeys
exihibited high amount of phenolics but it is necessary in future to determine TPC of isolated
samples by Folin-Cioucalteu method and HPLC. Comparison of TPC of honeys produced by
Apis florea (D) and Apis dorsata (G) is shown in Fig. 4.30. TPC of honey of Apis florea were
higher as compared to honeys produced by Apis dorsata. Phenolic contents of Pakistani
honeys were determined with the help of Folin-Ciocalteu method due to its simplicity and
rapidity but there may be interferences caused by ascorbic acid, free amino acids and
reducing sugars which may lead to the higher values than actual (George et al., 2005;
Ferreira et al., 2009). As the Folin-Ciocalteu method is not the final strategy for
quantification of phenolic contents hence other available protocols may also be opted for
comprehensive analysis.
99
99.33
65.41
0.00
20.00
40.00
60.00
80.00
100.00
120.00
D G
TPC
(m
g/ 1
00
g)
Honey bee Species
Fig. 4.29: Gallic acid equivalent phenolic contents of commercial honeys of Pakistan
Fig. 4.30: Total phenolic contents of natural honey produced by Apis florea (D) and Apis
dorsata (G)
4.2.2 Total flavonoid contents (TFC)
Table 4.7 showed the result of analysis of variance for extraction of flavonoids by
three solvents ethanol, water and water ethanol (50:50) mixture. Water and ethanol mixture
was most effective in extraction of flavonoids as shown in Fig. 4.31, 4.32 and 4.34. Yellow
bars represented flavonoids extracted by ethanol (100%) while green bars represented water
(100%) and purple bars indicate ethanol water (1:1). Mixture of ethanol and water were
21.01
89.81
1.328 14.72
27.3
48.35
7.5 0
25.48
58.92
137.54
211.28
3.28
140.55
3.34 0
50
100
150
200
250
A N B1 B2 B3 B4 B5 B6 IS1 IS2 S M C L H
TPC
(m
g/ 1
00
g)
Commercial Samples
100
found to extract maximum amount of flavonoids as indicated by purple bars on the other
hand water was found to exhibit minimum potential to extract flavonoids among all solvents
used in this study. Total flavonoid content of the Burkina Fasan honeys were in the range of
0.17±0.07 to 8.35±0.16 mg QE /100 g with a mean value of 2.57±2.09 mg QE/100 g (Meda
et al., 2005). Natural Pakistani honeys were found to have higher flavonoid contents as
compared to many reported honeys but results are comparable to Portugal honeys.
Comparison of TFC of different localities showed that honeys from Bahawalpur and Karror
have maximum values of flavonoids as shown in Fig. 4.31. Total flavonoid contents of
honeys from Shorkot and Abdulhakeem were found minimum among all the natural honeys.
Mean flavonoid contents of natural honeys of different localities were 36.48 mg/100 g.
Portugal honeys contained 123.62±0.17 to 587.42±0.46 mg/kg flavonoids (Ferreira et al.,
2009).
Table 4.8: Result of analysis of variance for extraction of flavonoids by different
solvents
Types of Honey samples P-Values R2
Abdulhakeem 0.00 99.90
Bahawalpur 0.00 99.90
Dunya Pur 0.00 100.0
Faisalabad 0.00 100.0
Layyah 0.00 99.95
Multan 0.00 100.0
Shorkot 0.00 98.78
Karror 0.00 100.0
Beekeeper‟s Honeys 0.001 58.52
Commercial Honeys 0.00 74.09
101
12.18 12.35 12.89 12.32 12.83 12.88 10.89 9.51
10.72
10.72 10.68 10.60 10.77 10.85
10.85 10.72 20.78
60.15
37.40
31.38 33.40 33.46
13.40
61.84
0
10
20
30
40
50
60
70 Ethanol
Water
Et:wat.
TFC
mg
/10
0 g
Sampling Area
Fig. 4.31: Total flavonoids (g/100 g) of natural honeys from different locations
Fig. 4.32: Total flavonoids contents (g/100 g) of honeys collected form beekeepers
6.44
11.98
6.12 6.21 6.71 6.16 6.08 6.26
4.22
10.74
4.36
4.74
4.58 4.43
4.57 4.46
8.01
36.48
8.54 10.48
5.53
8.78 6.87
5.43
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
Adultrated NaturalMean
B1 B2 B3 B4 B5 B6
Ethanol
Water
Et:wat.
TFC
(m
g /1
00
g)
Beekeeper's Honey Samples
102
Fig. 4.33: Total flavonoids contents g/100 g in honeys of different brands
Fig. 4.34: Total flavonoids (g /100 g) of honeys produced by Apis florea (D) and Apis
dorsata (G)
Total flavonoids contents of Romanian honeys were 0.91 to 28.25 mg/100 g (Al et
al., 2009). Total flavonoids in Venezuelian honeys were 2.32±1.09 to 14.41±3.62 mg EQ/
100 g (Vit et al., 2009). TFC values of beekeeper‟s honeys were much lower than natural
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
Ethanol
Water
Et:wat.
TFC
(m
g /1
00
g)
Honey Samples of different brands
11.67 10.89 13.68
12.32 10.59
31.38
0
5
10
15
20
25
30
35
40
45
water Ethanol W:E
D
G
TFC
(m
g /1
00
g)
Honey bee Species
103
honeys as shown in Fig. 4.32.Values of flavonoids in beekeeper‟s honeys were close to those
for adulterated honeys. Flavonoid contents of commercial honeys of different brands are
given in the Fig. 4.33. Flavonoid contents of branded honeys were also much less than our
natural honeys of different locations and close to adulterated honeys. Comparison of
flavonoid contents of honeys produced by Apis dorsata and Apis florea is shown in Fig. 4.34
as green and blue bars, respectively. Apis floreal honeys exihibited higher flavonoid contents
as compared to honeys produced by Apis dorsata.
4.2.3 Vitamin C. contents
Amount of vitamin C in natural Pakistani honeys was in the range of 125.22±15.71 to
231.01±11.78 mg/kg as shown in the Fig. 4.35. Natural honeys from Faisalabad, Multan and
Karor region were contained high amounts of vitamin C. Range of vitamin C contents in
Portugal honeys is 140.01±0.05 to145.80±0.02 mg/kg (Ferreira et al., 2009). Vit et al.,
(2009) analyzed nine honey samples from different locations of Venezuela. Venezuelean
Fig. 4.35: Vitamin C contents in natural Pakistani honey samples of different location
177.39 185.47
155.65
193.33
125.22
230.19
144.78
231.01
0
50
100
150
200
250
300
Vit
amin
C (
mg/
Kg)
Areas of Samples
104
83.31
122.36 128.87
63.00
119.32
90.81
107.34
94.78
86.09
96.63
87.54
0
20
40
60
80
100
120
140
IS1 IS2 Sa Ma Ch Mu La Sw Yo Li H
Honey Samples of different Brands
Fig. 4.36: Vitamin C contents in honey samples of some local brands available in
markets of Pakistan
4.37: Vitamin C contents in honey samples collected from some beekeepers of Pakistan
109.87 115.23
86.09 89.56
60.51
73.81
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
B1 B2 B3 B4 B5 B6Beekeeper's Honey Samples
105
148.41
197.68
0.00
50.00
100.00
150.00
200.00
250.00
G DHoney bee Species
Vit
amin
C (
mg/
Kg)
Honeys contained 120.86±0.23 to 370.05±9.90 mg/kg with mean value 220.04±7.35 mg/100
g while monofloral Italian honeys exhibited no ascorbic acid (Alvarez-Suarez et al., 2010).
Many natural honey samples from Pakistan contained much higher amount of vitamin C as
compared to Portugal honeys while our results were comparable to many Venezuelan
honeys. Some Venezuelan honeys contained more vitamin C as compared to natural
Pakistani honeys. Vitamin C contents in commercial honey samples of different brands were
in the range of 63.00±0.32 to 128.87±0.65 mg/kg as shown in Fig.4.36. Amount of vitamin C
in our beekeeper‟s honey samples was less than natural honey samples which were in the
range of 60.51±0.30 to 115.23±0.58 mg/kg as shown in Fig. 4.37.
Fig. 4.38: Vitamin C contents in honey samples produced by Apis florea (D) and Apis
dorsata (G)
Branded honey samples were also contained less amount of vitamin C as compared to
natural honey samples while honey samples of Islamic shahed centre (IS2), Salman‟s honey
(Sa), China honey (Ch) and Langanese honey (Lan) were higher as compared to other brands.
Honeys produced by Apis florea (D) contain more vitamin C content as compared to honeys
produced by Apis dorsata (G) as shown in Fig.4.38.
4.2.4 β-carotene
Beta-carotene concentrations of analyzed natural Pakistani honeys are shown in Fig.
4.39. β-carotene measured in natural honeys was found in the range of 23.11±12.74 to
106
130.96±13.64 mg/kg. Carotene in natural Pakistani honeys was much higher than Portugal
honeys. Carotene contents of beekeepers and branded honeys were much less than natural
honeys. All honey samples of beekeeper‟s were found to have zero carotenoid except two
honey samples which exhibited 10.22±0.00 and 11.56±0.89 mg/kg of carotenoids as shown
in Fig. 4.40. In case of our branded honey samples carotenoid contents were zero for many
Fig. 4.39: β- Carotene contents of natural honeys
Fig. 4.40: β- Carotene contents of commercial honey collected from beekeepers
5.21
80.16
11.56 10.22 0.00 0.00 0.00 0.00
0
20
40
60
80
100
120
140
A N B1 B2 B3 B4 B5 B6
Beekeeper's Honey samples
β-c
aro
ten
e (
mg/
Kg)
44.74
130.96
93.59 83.02
23.11
99.37
45.44
121.06
0
20
40
60
80
100
120
140
160
β
-car
ote
ne
(m
g/ K
g)
Sampling Area
107
Fig. 4.41: β- Carotene contents of honeys of some commercial brands
Fig. 4.42: β- Carotene contents of honeys produced by Apis florea (D) and Apis dorsata
(G)
samples as shown in Fig. 4.41. Swat honey, Young‟s honey, Langanese honey and Honey
contained 1.33±1.02, 9.63±3.56, 9.78±1.33 and 6.67±1.11 carotenoids. Carotenoid measured
in Italian (1.17±1.22 to 5.57±1.02 mg/kg), Portugal (8.64±0.06 to 9.49±0.15 mg/kg) (Ferreira
5.21
80.16
0 0 0 0 0 1.33 9.63 9.78 6.67
0
20
40
60
80
100
120
140
β-c
aro
ten
e (
mg/
Kg)
36.70
83.02
0.00
20.00
40.00
60.00
80.00
100.00
120.00
D G
β-c
aro
ten
e (
mg/
Kg)
Honey bee Species
108
2.79
2.49
1.48
0.94 1.54
1.81
7.80
4.20
3.06 3.31
3.47
3.23
0.55
2.27
3.15
3.44 3.35
2.82 2.36
1.05
2.20 1.93 1.25
0.54
0.79
0
2
4
6
8
10
12
Ab
du
lhak
e…
Bah
awal
pu
r
Du
nya
Pu
r
Fais
alab
ad
Layy
ah
Mu
ltan
Sho
rko
t
Kar
ror
B1
B2
B3
B4
B5
B6
Is1
IS2 Sa Ma
Ch
Mu La Sw
Y L C
Lyc
op
en
e (
mg/
Kg)
Honey Samples
et al., 2009) were less than our natural honey samples but comparable to some of our branded
honey samples. Mean carotenoid values of our honeys produced by Apis florae
(36.70±16.88) were less than Apis dorsata honeys (83.02±20.29 mg/kg) as shown in Fig.
4.42.
4.2.5 Lycopene contents
Lycopene values of studied Pakistani honeys are shown in Fig. 4.43. Lycopene concentration
Fig. 4.43: Lycopene contents of studied Pakistani honeys
of Pakistani honeys was found in the range 0.54 to 7.80 mg/kg. Samples collected from
Shorkot exhibited maximum lycopene level with a mean value of 7.80±2.50 mg/kg. All other
natural samples contained less than 4.20 mg/kg of lycopene. Minimum lycopene contents
among all natural samples were found for honeys of Faisalabad. Mean lycopene
concentration of honeys of Faisalabad was 0.94 mg/kg. Lycopene contents of commercial
honey samples were less as compared to natural honey samples. Portugal honeys were found
to reveal (6.12±0.03 to 6.55±0.01 mg/kg) of lycopene (Ferreira et al., 2009). Most of natural
honey samples exhibited lycopene comparable to Portugal honeys.
109
2.65
0.94
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
D GHoney bee Species
L
yco
pen
e (
mg/
Kg)
Fig. 4.44: Lycopene contents of Pakistani honeys produced by Apis florea (D) and Apis
dorsata (G)
Mean lycopene concentration of honeys produced by Apis florea and Apis dorsata
was 2.65±1.39 and 0.94±0.52, respectively. Therefore, Apis floreal were found to be richer in
lycopenes as compared to honeys of Apis dorsata as shown in Fig 4.44.
4.3 Antioxidant profile
Ascorbic acid equivalent and qurecitin equivalent antioxidant contents of all honey
samples were determined. DPPH and peroxide scavenging power and ferric reducing power
of all honey samples were also determined.
4.3.1 Total antioxidant contents (TC)
Comparison of AEAC and QEAC provide information about accuracy of results
because one molecule of ascorbic acid has two reaction sites for free radicals while one
molecule of qurecitin has only one reaction site for free radicals. In case of this study almost
all honeys have ratios comparable to the results found by Meda et al. (2005) for Burkina
Fasan (South Africa) honeys. TAC contents determined as AEAC and QEAC of our honey
samples have shown ratios approximately equal to 2 as found in reported honey samples. In
case of natural Pakistani honeys the values of AEAC and QEAC were found in the range of
8.30±0.55 to 22.10±1.47 and 2.85±0.17 to 14.64±0.86 mg/100 g of honey, respectively as
shown in
110
Fig. 4.45: Ascobic acid and qurecitin equivalent antioxidant profile of natural honeys
from different locations
Fig. 4.46: Ascobic acid and qurecitin equivalent antioxidant profile of commercial
honeys, adulterated honey and mean of natural honeys
13.54
18.03 17.19
15.83
20.35 19.17
14.93
18.66
7.26
14.91
10.40 9.22
13.13 12.11
8.46
11.65
0
5
10
15
20
25AEAC
QEAC
Tota
l an
tio
xod
ant
con
ten
ts (
g/1
00
g)
Sampling Areas
0.00
5.00
10.00
15.00
20.00
25.00
Tota
l an
tio
xod
ant
con
ten
ts (
g/1
00
g)
Commercial Honey Samples
111
Fig. 4.45. Commercial honeys have low % RSA and antioxidant values as compared to most
of natural Pakistani honeys honeys as shown in Fig. 4.46. Only four honeys have %RSA
more than 50%, on the other hand thirteen commercial honey samples have less than 50%
RSA. The same trend was found in the the case of ascorbic acid equivalent antioxidant
contents and qurecitin equivalent antioxidant contents of commercial Pakistani honeys.
AEAC and QEAC total antioxidant contents of Burkina Fasan were in the range of 10.20±
0.59 to 65.85± 0.010 and 4.27± 0.03 to 33.34±0.21 mg/100 g of honey, respectively (Meda et
al., 2005). Total antioxidant contents determined by Saxena et al. (2010) for seven Indian
honeys were in the range of 15.1± 0.7 to 29.5±1.80 mg AEAC/100 g honey. Most of Indian
honeys exhibited more than 50% RSA values. Antioxidant profile of Pakistani honeys is
comparable to Indian honey. This may be due geographical resemblance.
Isla et al. (2011) analyzed antioxidant contents of thirteen Argentinian honeys. They
found that maximum TAC values of Argentinian honeys were 10 µg/mL and 2.73 µg/mL
through DPPH and ABTS method, respectively. AEAC contents of Georgian honeys
determined by Taormina et al. (2001) were ranging from 139.9 to 1131.3 µM. Antioxidant
profile of forty Czech Republic honeys was evaluated by Lachman et al (2010) employing
FRAP, DPPH and ABTS method.
Fig. 4.47: Ascobic acid and qurecitin equivalent antioxidant profile of Apis florea
(Dwaf) and Apis dorsata (Giant)
0.00
5.00
10.00
15.00
20.00
25.00
Dwarf bee Giant BeeTota
l an
tio
xod
ant
con
ten
ts (
g/1
00
g)
Honey bee Specie
112
Results determined for different honeys are 98.73 to 441.98, 431.38 to 1026.38 and 222.98 to
887.12 by DPPH, ABTS and FRAP methods, respectively. Comparison of antioxidant
measurements of honeys produced by Apis florea and Apis dorsata are given in Fig. 4.47.
Antioxidant contents of honeys produced by Apis florea were higher as compared to Apis
dorsata.
4.3.2 Free radical scavenging activities
4.3.2.1 DPPH scavenging activity
Natural Pakistani honeys exhibited good antioxidant potential as compared to
available international data. Fig. 4.48 showed values of % radical scavenging activity
determined by DPPH assay for natural honeys used in our study. All samples collected from
Layyah, Dunyapur, Multan, Shorkot, and Karror have shown more than 50% inhibition.
While % RSA of ascorbic acid used as standard in our study was 77.92±1.45. Most of
samples from Bahawalpur and Faisalabad have also exhibited more than 50% inhibition.
Only one sample from Faisalabad out of twelve samples and one sample from Bahawalpur
out of seven has scavenged less than 50% RSA. % RSA of natural honeys is in the range of
30.50±0.31 to 77.43±0.77 with good mean value 60.41±11.63. RSA (%) of twenty four
Romanian honey samples determined by Al et al. (2009) is from 35.80% to 64.83% while %
DPPH radical scavenging activity of fifty rhododendron honeys of Turkey is from
36.11±8.27 to 90.73±0.00. Turkish honeys with excellent antioxidant potential were also
effective against certain bacteria (Silici et al., 2010). In our study, most of Pakistani honeys
showed more than 50% RSA.
Antioxidant results of seventy Slovenian honeys reported by Bertoncelj et al. (2007)
predicts that mean IC50 of different honeys are 7.2± 1.2 to 53.8 mg/mL by DPPH method
while FRAP values were in the range of 71.0±10.2 to 478.5±95.5 µM. Ferreira et al. (2009)
evaluated antioxidant profile of three Portugal honeys. They found that EC50 of DPPH
inhibition was from 106.67±2.48 to 168.94±19.20 mg/mL and reducing power was in the
range of 13.26±0.20 to 48.95±1.61 mg/mL. Honey samples of Italy have IC50 values 5.0 ±
15.5 ± 0.8 mg/mL (Buratti et al., 2007).
113
Fig. 4.48: Radical scavenging activity of natural honeys from different locations
Fig. 4.49: Radical scavanging activity of standard, adulterated, mean of natural and
commercial honey samples
IC50 of honey samples of Burkina Fasan was in the range of 1.37 ± 0.03 to 29.13 ± 1.50
(Meda et al., 2005). Fig. 4.48 shows the comparison of means RSA (%) among different
77.92
48.31
63.23 60.79 56.17
71.37 67.38
53.14
65.71
0
10
20
30
40
50
60
70
80
90
in
hib
itio
n o
f D
PP
H r
adic
al (
%)
Sampling Areas
77.92
42.105
60.76 57.14
51.12
33.09
66.57
47.56
23.37
49.6
41.52
33.68
40.55 44.85
53.48
60.81
0
10
20
30
40
50
60
70
80
90
inh
ibit
ion
of
DP
PH
rad
ical
(%
)
Commercial Honey Samples
114
types of Pakistani honeys from all natural localities. Among natural samples mean of % RSA
for samples of Layyah was excellent (71.37%). Mean values of RSA (%) of honey samples
of Multan, Karror, Bahawalpur and Dunyapur were also above 60%. First bar in Fig. 4.48
and 4.49 indicated the RSA (%) of ascorbic acid used as standard in this study. In case of
commercial Pakistani honey, %RSA values as shown in Fig.4.49 are also quite good for
many honeys. One beekper‟s sample has more than 60% RSA whereas two samples have
showed more than 50% RSA. % RSA of honeys available with name of honey and langanase
honey were more than 50% while all other samples have less than 50%. Therefore, branded
honeys have showed low %RSA values which may be due to adulteration. Overall, all
Pakistani honeys have good radical scavenging activities. Comparison of %RSA of honeys
produced by Apis florea and Apis dorsata is shown in Fig. 4.50. Both types of honeys were
collected from same locality. Mean of %RSA of honeys produced by Apis florae is little bit
higher than honey produced by Apis dorsata. There is not too much difference of %RSA
values among both types of honeys. This may be due to similarity in locality of sampling.
%RSA of honeys is due to bioactive compounds which come from floral sources instead of
honey bee this is why both types has similar %RSA values (Al et al., 2009).
Fig. 4.50: RSA (%) of honey samples produced by Apis florea (D) and Apis dorsata (G)
59.89 56.17
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
D G
inh
ibit
ion
of
DP
PH
rad
ical
(%
)
115
4.3.2.2 Ferric reducing antioxidant power (FRAP)
Ferric reducing antioxidant power (FRAP) of natural Pakistani honeys from different
localities is given in Fig. 4.51. FRAP values of natural honeys were found to be in the
805.96±10.47µM for beekeeper‟s and branded honeys, respectively while mean FRAP value
for adulterated honeys was 294.43±14.42 µM as shown in Fig. 4.52. Beekeeper‟s honey and
Young‟s, Swat, China, Islamic and Cooperative honey samples exhibited lower FRAP values
then natural Pakistani honeys while other branded honeys exihibited near to lower range of
natural Pakistani natural honeys. Beekeeper‟s honey samples exihibited FRAP values near to
adulterated honey samples. Mean FRAP values of Apis dorsta was much higher than Apis
florea as shown in Fig. 4.53. Bertoncelj et al. (2007) analyzed seventy honey samples of
Slovenia. FRAP values of Slovenian honeys were 71.0±10.20 to 478.5 ± 95.50 µM. FRAP
values for eighteen multifocal and unifloral honeys of Italy were from 61.75 to 124.50 µM
(Blasa et al. 2007). Unifloral honeys has shown less FRAP values than multifloral honeys.
Taormina et al. (2001) determined FRAP values of six Georgian honeys which were found in
the range of 120.30 to 1353.50 µM.
Fig. 4.51: Ferric reducing antioxidant power of natural Pakistani honeys from different
localities
435.09
1200.74
770.96
637.84
819.43
976.39
447.26
837.13
0
200
400
600
800
1000
1200
1400
Sampling Areas
FR
AP
(µ
M)
116
Fig. 4.52: Ferric reducing antioxidant power of commercial Pakistani honeys
range of 435.09± 68.33 to 1200.74±117.48 µM. In case of commercial Pakistani honeys
FRAP values were in the range of 112.91±1.47 to 445.96±5.79 and 193.78±2.52 to
Fig. 4.53: Ferric reducing antioxidant power of Apis florea and Apis dorsata
4.3.2.3 Peroxide scavenging activity
Peroxide scavenging activity of honey is due to presence of glucose oxidase added by
honey bee. All Pakistani honeys have good peroxide scavenging activity with more than 50%
294.43
445.96 396.39
192.48
112.91 132.91
119.43
449.00
193.78
344.65
464.22
222.91
537.70
425.96 462.91
325.96
805.96
314.21
0
100
200
300
400
500
600
700
800
900
FRA
P (
µM
)
Commercial Honey Samples
446.54
637.84
0
100
200
300
400
500
600
700
800
Apis florea Apis dorsata
Honey bee Species
FR
AP
(µ
M)
117
peroxide inhibition. Peroxine inhibition of natural Pakistani honeys was in the range of
57.37±2.08 to 71.69±9.74 as shown in Fig. 4.54. Natural Pakistani honeys from different
Fig. 4.54: Inhibition (%) of peroxide by natural honeys from different locations
localities have close peroxide inhibition values. Most of the honeys have % peroxide
inhibition more than 60%. Peroxide inhibition values of commercial Pakistani honeys are
given in Fig. 4.55. Range of peroxide inhibition was 45.54±0.76 to 66.13±1.11%. Peroxide
inhibition values for beekeeper‟s honeys were less as compared to other commercial honeys
and natural honeys. Peroxide inhibition (%) of ascorbic acid (0.1 mg/mL) used as standard
was equal to 63.46±.03%. Presence of catalase is reported by many authors which is
responsible for peroxide inhibition. Catalase has one of the highest turnover numbers and can
66.24 62.80
58.67
71.69
62.08 60.71 61.28 62.78
57.37
0
10
20
30
40
50
60
70
80
90
inh
ibit
ion
of
DP
PH
rad
ical
(%
)
Sampling Areas
118
Fig. 4.55: Peroxide inhibition (%) of standard, adulterated, mean of natural samples
and commercial samples
convert millions of hydrogen peroxide molecules to water and oxygen in a second. Catalase
can be use to remove hydrogen peroxide from different products. Honey consumption is
helpful to increase the antioxidant and reducing potential of human plasma (Saxena et al.,
2010). Peroxide inhibition of honeys produced by Apis dorsata and Apis florea were almost
equal as shown in Fig. 4.56.
66.24
55.69
62.17
51.74
55.83
45.54
55.30 53.94
50.06
63.67 64.27 63.17
62.87
64.47
63.32
63.13 66.53
63.46 63.01
62.12
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
inh
ibit
ion
of
DP
PH
rad
ical
(%
)
Commercial honey samples
119
Fig. 4.56: Peroxide inhibition (%) of honey samples produced by Apis florea (D) and
Apis dorsata (G)
4.4 Antimicrobial activities
4.4.1 Antibacterial activities
Many studies on different types of honeys in different origins of world by many
researchers provided strong evidence in support of antimicrobial potential of honey (Alvarez-
Suarez et al., 2010; Gulfraz et al., 2010; Lee et al., 2008). Bioactive components (from floral
sources and originated due to honey bee processing), acidic behavior and low moisture
contents were reported as factors responsible for antimicrobial properties of honey. All
analyzed natural Pakistani honeys follow international standards of honeys. Mean zones of
inhibition shown by analyzed Pakistani honey samples against Gram positive and Gram
negative bacteria were given in Table 4.9. Natural honey samples collected from
Abdulhakeem, Bahawalpur, Dunya Pur, Faisalabad, Layyah, Multan, Shorkot and Karror
have shown mean zones of inhibition against Pasteurella multocida in the range of 14 to 22,
16 to 20, 12 to 22, 18 to 22, 16 to 22, 12 to 24, 12 and 14 to 22 mm, respectively. Beekeepers
and many branded honey samples have shown no zones against Pasteurella multocida as
indicated by blanks (-). Some commercial honeys available as brand names of Muqeet
Honey, Langanese Honey, Swat honey and Young´s honey have exhibited zones of inhibition
62.19 62.08
58.00
59.00
60.00
61.00
62.00
63.00
64.00
65.00
D G
inh
ibit
ion
of
DP
PH
rad
ical
(%
)
Honey bee species
120
Table 4.9: Mean zone of inhibition (mm) shown by honeys against Gram positive and
negative bacterial strains
Continued........
Locality of Samples Pasteurella
multocida
Escherichia
coli
Bacillus
subtilus
Staphylococc
us aureus
Abdulhakeem 16 22 14 10
16 22 14 14
22 20 20 12
14 20 20 10
Bahawalpur 10 10 12 10
16 24 20 22
16 16 24 22
20 10 24 14
16 12 24 12
16 20 18 14
18 14 22 22
Dunya Pur 22 20 12 20
14 16 12 10
12 22 20 16
12 20 20 22
Faisalabad (Dwarf) 12 10 10 16
14 14 22 24
14 16 20 22
20 16 18 12
16 10 16 10
10 18 12 10
Giant 18 10 16 16
22 22 12 22
121
Continued.................
Locality of Sample Pasteurella
multocida
Escherichia
coli
Bacillus
subtilus
Staphylococc
us aureus
10 18 18 10
18 12 16 10
18 12 14 10
22 24 16 20
Layyah 16 20 20 12
22 18 22 14
Multan 24 24 12 16
14 14 18 12
16 24 18 14
20 16 14 16
24 18 20 20
24 20 20 22
12 14 22 16
20 14 20 16
18 26 22 10
Shorkot 12 12 14 16
12 12 10 16
10 20 12 18
10 24 12 10
Karror 14 28 14 16
16 20 22 14
22 18 20 12
Beekeepers - - 16 20
- - 16 12
- 18 16 -
- 18 18 24
122
The values given in the table are the mean of three independent experimental repeats
of 14 mm diameter. Overall, natural Pakistani honeys developed zones of inhibition in the
range of 10 to 28 mm for all tested bacterial strains. Honey samples of Abdulhakeem,
Bahawalpur, Dunya Pur, Faisalabad, Layyah, Multan, Shorkot and Karror have shown mean
zones of inhibition against Escherichia coli were in the range of 20 to 22, 10 to 24, 16 to 22,
10 to 24, 18 to 22, 14 to 26, 12 to 24 and 18 to 28 mm, respectively. Only two honey samples
of beekeepers and all branded honey samples have shown poor inhibitory action against
Escherichia coli. All other beekeeper‟s honey samples and one branded sample have no
inhibition against Escherichia coli. Honey samples of Abdulhakeem, Bahawalpur, Dunya
Pur, Faisalabad, Layyah, Multan, Shorkot and Karror have shown mean zones of inhibition
against gram positive bacteria Bacillus subtilus and Staphylococcus aureus were in the range
of 14 to 20 and 10 to 14, 12 to 24 and 10 to 22, 12 to 20 and 16 to 22, 10 to 22 and 10 to 24,
20 to 22 and 12 to 14, 12 to 22 and 12 to 22, 10 to 14 and 10 to 18 and 14 to 22 and 12 to 16
mm, respectively. Commercial honey samples except some honey samples have shown poor
inhibitory action. Zones of inhibitions produced by different bacterial strains by some honey
Locality of Sample Pasteurella
multocida
Escherichia
coli
Bacillus
subtilus
Staphylococcu
s aureus
- - 16 -
- - 14 -
Islamic Shahed - 20 20 20
- 20 16 22
Salman´s Honey - 16 16 14
Marhaba honey - 14 14 16
China Honey - 12 14 -
Muqeet Honey 14 14 24 -
Langanese Honey 14 14 22 16
Swat honey 14 14 14 16
Young´s honey 14 12 12 -
Life Style - 22 14 12
Honey - - 18 26
Standard 28 30 24 28
123
samples are given in the Fig 4.57a while Fig. 4.57b represents the growth areas produced
against the beekeepers honey samples.
I II
III IV
Fig. 4.57a: Zones of inhibition of some honeys against Pasteurella multocida (I), zones of
inhibition of some honeys against Escherichia coli (II), Bacillus subtili (III) and
Staphylococcus aureus (IV)
124
Fig. 4.57b: Pasteurella multocida growth produced by some beekeeper’s honeys
A study conducted by Alvarez-Suarez et al. (2010) antimicrobial action of honey was
found more effective against gram positive bacteria as compared to gram negative bacteria.
Different types of floral honeys were found to exhibit poor inhibitory action against S.
aureus (1.83 to 4.78), E. coli (6.13 to 12.48) and B. subtilus (6.88 to 10.93 mm) as compared
to our natural honey samples (Manyi-Loh et al., 2010). Honey formulations were also found
to have less inhibition (0.51±0.1 to 2.91±0.2, 0.72±0.1 to 3.12±0.9, 1±0.4 to 3.51±0.6 and
0.92±0.5 to 3.45±0.7 mm for S. aureus, E. coli, Candida albicans and Aspergillus niger,
respectively) as compared to the pure honey (Gulfraz et al., 2010). Irish honeys from
mountain regions have also shown excellent inhibitory action comparable to our honey
samples against Staphylococcus aureus species (Maeda et al., 2008). Mean zones of
inhibition of apiarist‟s honeys and of Argentina (17.1±0.1 and 13.2±0.1 mm) were also
comparable to our results. Pure honey samples of apiarist and honey packer‟s possessed
excellent antimicrobial potential in the range of 15 to 24 and 5 to 15 mm, respectively against
S.aureus, S. epidermidis, M. luteus, S. uberis, E. faecalis, E. coli, K. pnemoniae and P.
aeroginosa (Basualdo et al., 2007). In the following study apiarist honey samples exhibited
inhibitory potential close to our natural honey samples.
4.4.2 Minimum inhibitory concentrations
Minimum inhibitory concentrations to resist 50% of microbial growth are given in
Table 4.10. MIC50 of natural Pakistani honeys were in the range of 1.18±0.02 to 15.73±0.22,
5.44±0.53 to 16.66±0.67, 2.23±0.04 to 13.89±1.30 and 2.63±0.01 to 13.45±0.69 %V/V for
125
Table 4.10: Mean MIC50 values of honeys samples V/V (%) against Gram positive and
Gram negative bacteria
Locality of Sample Bacillus
subtilus
Staphylococcus
aureus
Escherichia coli Pasteurella
multocida
Abdul Hakeem 10.97±0.15 15.22±0.46 6.96±1.22 5.35±0.70
11.63±0.30 10.60±0.21 5.54±0.97 7.02±0.91
11.81±0.73 11.80±0.42 13.89±1.30 8.33±1.04
5.02±0.63 6.08±0.61 4.16±0.07 5.00±0.00
Bahawalpur 4.00±0.61 6.49±0.61 3.45±0.06 4.3±0.01
6.36±0.96 6.29±0.66 3.49±0.06 8.05±1.05
5.77±0.74 7.40±0.68 4.22±0.07 4.47±0.58
12.53±0.20 8.59±0.09 3.63±0.06 9.04±0.77
11.87±0.59 6.42±0.65 3.93±0.07 9.14±0.90
11.09±0.90 10.64±0.51 10.93±0.56 6.82±0.83
7.55±0.25 11.60±0.22 7.60±1.19 4.91±0.53
Dunya Pur 1.18±0.02 7.94±0.38 9.18±1.39 6.23±0.82
8.38±-0.12 6.24±0.57 6.86±1.12 4.12±0.53
5.14±0.07 13.74±0.14 3.55±0.06 6.93±0.48
6.40±0.65 5.50±0.46 7.05±0.83 6.03±0.84
Faisalabad(Dwarf) 5.31±0.50 6.30±0.47 6.97±1.17 7.72±0.62
5.79±0.74 8.02±0.69 7.38±1.34 8.61±1.07
4.79±0.60 16.06±0.65 10.64±0.71 7.77±0.97
7.94±0.68 7.35±0.74 5.03±0.95 7.94±0.62
6.27±0.92 10.59±0.51 11.10±1.25 11.11±0.40
6.83±0.24 11.55±0.42 4.56±0.43 4.85±0.53
Faisalabad (giant) 4.97±0.07 7.65±0.71 6.44±1.13 7.66±0.23
9.55±0.46 11.63±0.36 12.05±0.44 10.37±0.39
7.30±0.53 11.30±0.72 11.39±1.25 13.75±0.83
6.08±0.51 7.52±0.72 11.86±3.72 4.23±0.05
8.07±0.68 8.19±0.39 8.61±1.38 11.76±0.15
Continued…………
126
Locality of Sample Bacillus
subtilus±SD
Staphylococcus
aureus±SD.
Escherichia
coli±SD.
Pasteurella
multocida±SD.
7.14±0.66 9.70±0.60 9.48±1.22 7.24±0.91
Layyah 5.04±0.78 6.56±0.61 7.05±1.00 7.85±0.92
5.77±0.36 11.58±0.62 7.37±1.34 5.90±0.71
Multan 8.22±1.10 6.80±0.61 6.88±0.47 7.19±0.74
15.73±0.22 7.44±0.48 2.76±0.05 3.22±0.04
8.74±0.83 7.46±0.68 4.99±0.09 2.63±0.01
10.92±0.44 11.32±0.62 2.23±0.04 2.98±0.01
7.36±0.95 6.93±0.65 9.21±1.39 7.03±0.99
6.36±0.91 7.04±0.71 8.45±1.45 5.00±0.71
5.28±0.72 7.82±0.58 4.01±0.70 9.01±0.90
4.37±0.58 7.06±0.71 8.16±1.41 6.72±0.35
4.17±0.06 9.84±0.40 8.98±0.16 10.40±0.28
Shorkot 4.87±0.49 5.94±0.55 3.31±0.57 6.39±0.73
9.27±0.69 6.92±0.64 5.06±0.91 6.74±0.79
4.90±0.60 8.47±0.79 7.37±1.30 6.49±0.73
9.53±0.42 10.25±0.61 7.78±1.35 10.12±0.39
Karror 6.25±0.37 5.44±0.53 4.45±0.08 2.92±0.04
8.61±1.22 16.66±0.67 5.46±0.10 4.0±0.00
11.73±0.78 6.67±0.66 7.43±0.83 13.45±0.69
Beekeepers 44.90±0.63 71.18±0.72 53.25±0.93 70.78±0.92
39.30±0.55 42.13±0.43 31.6±0.06 62.99±0.82
60.08±0.09 44.23±0.45 3.67±0.06 60.79±0.79
28.92±0.41 19.13±0.19 31.7±0.06 24.43±0.32
25.17±0.35 45.09±0.46 49.04±0.86 27.01±0.35
67.73±0.95 5``0.93±0.51 40.0±0.07 47.38±0.62
Islamic Shahed 41.70±0.59 36.21±0.37 42.00±0.01 69.43±0.90
66.08±0.93 51.98±0.53 41.6±0.07 73.44±0.95
Salman´ Honey 63.92±0.90 53.55±0.54 40.9±0.07 40.62±0.53
Marhaba honey 54.45±0.77 82.36±0.83 37.2±0.07 66.40±0.86
Continued……..
127
The values given in the table are the mean of three independent experimental repeats
Bacillus subtilus, Staphylococcus aureus, Escherichia coli and Pasteurella multocida,
respectively. MIC50 of commercial honey samples were more than 50% V/V. Therefore,
natural Pakistani honeys have bioactive components and exhibited antibacterial potential due
to presence of antioxidant compounds even at dilutions. Minimum active dilution of Italian
honeys against Bac. Subtilus, S. aureus, P. aeroginosa and E. coli were in the range 4.7±0.5
to 10.40±0.70, 2.50±0.50 to 50±0.60, 7.20±0.50 to excellent MIC50 values at low honey
concentrations while commercial honeys have poor inhibition potential. In pure form
antibacterial action of honey is due to low pH and moisture 16±0.50 and 4.7±0.50 to
12.5±0.70 %V/V, respectively. Monofloral honeys were found to have more inhibitory
potential at diluted concentrations as compared to our honey samples (Alvarez-Suarez et al.,
2010). Indian honeys to inhibit were also found to excellent MIC values as found for our
honey samples (Mandal et al., 2010). Antibacterial potential of studied natural Pakistani
honeys were much higher as compared to many other types of honeys as reported in
literature. On the other hand, our commercial honey samples have shown poor activities as
compared to natural honeys. Contrary to the antimicrobial inhibition potential; honey is also
reported to contain some microbes like yeast and some spore forming bacteria (especially
Clostridium botulinum). It is also reported that honey is free from vegetative disease causing
bacteria due to inhibition potential. On the other hand presence of high number of bacteria
may be due to contamination and poor storage techniques (National Honey Board, 2005). So,
it is recommended to analyze the microbial load on electron microscopic level and the effect
microbial load on antimicrobial potential of honeys stored under different conditions.
Locality of Sample Bacillus
subtilus±SD
Staphylococcus
aureus±SD.
Escherichia
coli±SD.
Pasteurella
multocida±SD.
China Honey 48.68±0.69 71.05±0.72 3.66±0.06 53.56±0.70
Muqeet Honey 68.45±0.96 58.92±0.60 40.9±0.07 65.29±0.85
Langanese Honey 74.51±1.05 58.81±0.59 11.1±0.02 42.53±0.55
Swat honey 40.12±0.57 57.57±0.58 31.72±0.56 48.71±0.63
Young´s honey 69.04±0.97 78.67±0.79 66.34±1.16 39.96±0.52
Life Style 72.46±1.02 53.36±0.54 72.73±1.28 48.97±0.64
Cooperative 75.76±1.07 56.04±0.57 28.65±0.50 64.18±0.83
Standard 0.05±.01 0.05±.01 0.05±.01 0.05±.01
128
Isolated fractions of phenolics, flavonoids, ascorbic acid and other bioactive fractions should
be used to highlight the factors involve in the antimicrobial properties of honey.
4.4.3 Antifungal activity
Analyzed honey samples from Pakistan were not effective to inhibit the growth of all
tested fungal strains. Results obtained by spectrophotometric assay and disc diffusion method
indicate that natural, beekeepers and branded honey samples were not effective against all the
tested fungal strains
4.5 Antitumor activity of Pakistani honeys
Although no data is available for antitumor activity of honey against Agrobacterium
tumefaciens but extensive evidence about antimicrobial potential is availabe in literature
(Taormina et al., 2001; Kucuk et al., 2007; Silici et al., 2010; Brudzynski and Miotto, 2011;
Isla et al., 2011). We have analyzed our honey samples against wide range of microbes
which proved honey as potential natural product of bioactive importance. So, here we
compared antitumor activity of honeys with other potential antitumor natural products
Fig. 4.58: Petri plates of potato disk assay of some honey samples after twenty one days
of incubation
investigated by some authors. Fig. 4.58 shows the picture of a Petri dish taken after twenty
one days of incubation of potato disks fed by some honey samples with positive and negative
129
control in the center. Only two natural Pakistani honey samples out of forty five samples
have shown less than 20% inhibition. All other honey samples have shown excellent
antitumor potential with values more than 20% inhibition. Antitumor activities of
commercial honeys were also found quite well, values of thirteen samples were more than
20%. Sample number five and seven from Faisalabad have shown antitumor activity more
than standard; vincristine sulphate (as shown in Fig.4.59 red bar for standard). Maximum
antitumor activity shown by a natural honeys collected from Faisalabad is 87.50±5.50% and
79.00±05.66 as indicated by green bars. All Natural honeys collected from Dunya pur have
also shown good antitumor activitiy values. All samples of Dunyapur have shown more than
35% and two sampleshas more than 50% inhibition. Both honey samples collected from
Fig. 4.59: Inhibition (%) of tumors by different types of Pakistani honeys
Layyah has shown more than 40% tumor inhibition as shown by orange bars in Fig. 4.59.
Red bar indicates the % inhibition caused by standard vincristine sulphate while other bars
0
20
40
60
80
100
120
Honey Samples
Tu
mo
r In
hib
itio
n (
%)
130
show % inhibition of different Pakistani honeys. Mazid et al. (2011) reported that different
extracts of polygonum barbatum and polygonum stagninum showed good antitumor activities
with IC50 from 180 to more than 400 µg/disc. Lower IC50 indicates the higher antitumor
potential. Mahamuni et al. (2012) analyzed fruits of Lagenaria siceraria proves that this is a
good plant for antitumor potential. Average numbers of tumor formation in presence of
Lagenaria siceraria extract (1000 ppm) of n-Butanol is only one as compared to simple
solvent which has 22.05 tumers. Methanolic and aqueus extracts of garlic (Allium stivum L.)
were analyzed by Hosseinzadeh et al. (2011) for their antitumor activity. Antitumor activities
of commercial Pakistani honeys were also good. Marhaba honey, China honey and Sulman‟s
honey has shown more than 50% tumor inhibition.
I II
III IV
Fig. 4.60: Tumor formation in disk of positive control (I), negative control (II), sample
with excellent tumor inhibition (III) and sample with no tumor inhibition (IV)
On the other hand some commercial honeys like Swat honey, Islamic honey and honey have
shown less than 20% tumor inhibition. Many Pakistani honeys have excellent antitumor
potential as compared to many plant extracts. Islam et al. (2011) of Bangladesh found that
131
croton bonplandianum is a potential antitumor drug. % tumor inhibition more than 35% in
the case of 1000 ppm solution of extracted plant material against three species of
agrobacterium. Jamil et al. (2012) analyzed some Pakistani plants for antitumor activity and
nearly all plants showed excellent % inhibition. Methanolic extract of Q. dilata showed
84.78% inhibition which proved it to be quite effective anticancer plant. % inhibition of most
of honeys is more than many natural products. Samples with more than 50% antitumor
activity also show more than 50% RSA. Peroxide (%) and more analyzed some Pakistani
plants for antitumor activity and nearly all plants show excellent % inhibition. Methanolic
extract of Q. dilata showed 84.78% inhibition which proves it very good anticancer plant.
Hadizadeh et al. (2007) determined % inhibition of their synthetic compounds. % inhibition
of one compound is -80.50% as compared to positive control which is only -67.24%. Fig.
4.60 shows the pictures of potato disks fed with positive control, negative control and two
different honey samples.
132
CHAPTER 5 SUMMARY
Honey is sweet natural product composed of constituents originated from plants and
salivary glands of honey bees. Internationally a lot of work has been done and reported on
the chemical and medicinal characterization of honeys. Honey is composed of amino acids,
sugars, aroma compounds, flavors, phenolic compounds, flavonoids, ascorbic acid, aliphatic
compounds, organic acids, norisoprenoids, terpenoids and minerals. Honey bees may carry
some components with its feet or body during its visits and so some components from
surrounding air, water and soil may also be added by honey bee. Analysis of
physicochemical properties of honey is helpful not only for quality evaluation but also for
characterization of botanical and geographical origins of honey. It is also useful to highlight
the adulteration. Honey is not only important due to its unique composition but also due to its
tremendous therapeutic properties. Phytochemical constituents, antioxidants potential, low
moisture contents and acidic behavior are the parameters responsible for antimicrobial,
therapeutic, anticancer and wound healing properties of honey. Present study was designed
for evaluation of chemical, nutritional and medicinal parameters of natural Pakistani honeys
from different locations and some commercially available honeys from Pakistan.
Adulteration of commercial honeys was detected by using comparison of these parameters as
quality tool. Toxicity of heavy metals in surrounding environment was also detected by
honey analysis while Appraisal of antimicrobial, antioxidant and anticancer activities of the
honeys were an objective of this study.
Electrical conductivity, pH, refractive index, ash and moisture (%) were 0.42±0.13 to
0.86±0.02 mS/cm, 3.81±0.1 to 3.87±0.06, 1.4845 to 1.4876, 0.23±0.001 to 0.80±0.001% and
17.80±0.28 to 19.67±0.81 for natural Pakistani honeys, 0.33±0.01 to 0.94±0.01 mS/cm,
3.61±0.0.02 to 4.24±0.03, 1.4848 to 1.4991, 0.01±0.00 to 0.20±0.000% and 15.20±1.22 to
20.60±1.58 for commercial Pakistani honeys analyzed in this study, respectively. EC values
of beekeeper‟s honey samples were closer to those found for natural Pakistani honeys.
Natural and commercial Pakistani honey samples contained 0.96±0.25 to 1.70±0.01 and 0.14
to 1.14 g/100 of protein. Pakistani honeys contained more protein as compared to available
133
international data. Natural, beekeeper‟s and branded honey samples exhibited 199.05±63.40
to 2336.91±45.37, 44±0.00 to 305±5.20 and 87.20±9.3 to 856.79±4.27 total amino acid and
129.30±0.82 to 661.80±4.49, 7.00±0.08 to 47.70±0.88 and 41.80±0.08 to 149.00±0.99,
respectively. Fructose, glucose and maltose found in the natural and beekeeper‟s honey
samples were in the range 25.75±0.25 to 37.33±1.77, 15.69±0.12 to 22.25±1.11 and
4.11±0.22 to 7.25±0.55% and 10.44±0.52 to 26.28±1.34, 11.28±0.56 to 25.26±1.25 and
0.16±0.01 to 9.55±0.48, respectively. Fructose glucose ratio of all natural honey samples
were more than 1.14. δ13
C values of all natural honey were in the range of -26.07 to -
27.0.14%. δ13
C values for all beekeeper‟ honey samples and many branded honey samples
were less than 23.5% which make them suspected and fulfill the statement of Pakistan
science foundation that most of commercial Pakistani honeys are adulterated. Fructose,
glucose and moisture were major components of our honeys. Fructose is major
monosaccharide of all natural honey samples and glucose is major monosaccharide of honey
samples of beekeeper‟s which is an indication of bee feeding with artificial sweeteners. This
fact was supported by isotopic ratio of C13
/C12
. Physicochemical characterizations of
Pakistani honeys place them at the level of international standards. Concentration of Ca, Mg,
Zn, Mn and Fe in analyzed honey samples were in agreement with international data. Cu
toxicity was found in many honeys of Bahawalpur, Multan and Karror regions whereas lead
toxicity was observed in honey some honey samples of Bahawalpur, Faisalabad and Multan
regions. Cobalt toxicity was found in honey samples of Bahawalpur, Multan, Faisalabad and
Abdulhakeem regions. Lead and cromium toxicity was found in many honey samples of
Abdulhakeem, Bahawalpur, Dunyapur, Multan, Faisalabad, Shorkot regions and many
commercial honeys as well. Bahawalpur, Dunyapur, Multan, Faisalabad regions were also
found to contain nickel toxicity. Almost no metal toxicity was found in our commercial
honeys. Total phenolic, total flavonoids, ascorbic acid, beta carotenoids and lycopene were
quantitatively found in natural honey samples in the range of 55.41 to 165.61 mg gallic acid
equivalent /100 g, 13.40 to 61.84 mg qurecitin equivalent /100 g, 125.22 to 231.01, 23.11 to
130.96 and 0.94 to 7.80 mg/ kg, respectively, on the other hand, all these components were
found in much less amount in commercial honey samples.
Samples from regions like Faisalabad, Multan, Bahawalpur, Abdulhakeem and
Dunyapur were found to contain toxic metals more than prescribe levels. Lead and chromium
134
toxicity was observed as more common. All studied honey samples have shown excellent
DPPH radical scavenging power, ferric reducing antioxidant power and peroxide scavenging
activities. Natural Pakistani honeys produced zones of inhibitionin the range of 10-28 mm for
all tested bacterial strains. Antifungal potential against all analyzed strains was very poor.
Many Pakistani honeys have excellent antitumor potential as compared to many plant
extracts. Some natural samples have antitumor activity close to the standard whereas two
samples have more than standard. Many commercial honey samples also have appreciable
antitumor activities. Overall, Pakistani honeys were promising natural food products with
excellent nutritional and medicinal properties.
135
Future prospective
Isolated Phenolics, flavonoids and other bioactive fractions of all honey samples
using amberlite columns and different combinations of solvents should be characterized for
qualitatively and quantitatively with the help of techniques like HPLC, LCMS and GCMS.
These isolated extracts should also be analyzed for their antimicrobial and anticancer
properties. These properties may then be correlated with properties analyzed for crude
honeys. Anticancer, ant diabetic and anti-hepatitus properties of these isolates and crude
honeys should be determined with the help of in vitro and in vivo trials. Classifications based
on the floral origin of Pakistani honey should be explored by identification of floral markers
like free amino acid, flavonoids and volatile compounds by techniques like amino acid
analyzer, GC, GC-MS and LC-MS. On the other hand detection of pesticide residues in
honey samples collected from agricultural and industrial areas should be carried out for
indication of environmental pollution.
136
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Glossary AOAC Association of Official Analytical Chemists
C3 Flowering exhibited the photosynthesis by Calvin–Benson cycle
C4 Corn and cane plants exhibited photosynthesis by Hatch-Slack
cycle
CODEX collection of internationally recognized standards, codes of
practice, guidelines and other recommendations relating to foods
DMPD N, N-dimethyl-p-phenylenediamine
DPPH 2, 2-diphenyl-1-picrylhydrazyl
EU European Union
FAO Food and Agriculture Organization
FRAP Ferric reducing antioxidant power
GC Gas chromatography
GCMS Liquid Chromatography-Mass Spectrometry
HFCS High fructose corn syrups
HMF hydroxylMethylfurfuraldehyde
HPLC High performance liquid chromatography
MIC Minimum inhibitory concentration
LCMS Liquid Chromatography-Mass Spectrometry
MRLPs maillard rection products
NDLUS Nutrient Data Laboratory of United States
ORAC Oxygen radical absorbance capacity
R Regression Analysis
TEAC Trolox equivalent antioxidant capacity
TFC Total Flavonoids Contents
TPC Total Phenolic Contents
USA United States of America
USDA United States Department of Agriculture
WHO World Health Organization
166
Annexure-I Relationship of moisture contents with refractive index values of honey
Refractive
Index (20ºC)
Moisture
Content
Refractive
Index (20ºC)
Moisture
Content
Refractive
Index (20ºC)
Moisture
Content
1.5044 13.0 1.4961 16.2 1.4880 19.4
1.5038 13.2 1.4956 16.4 1.4875 19.6
1.5033 13.4 1.4951 16.6 1.4870 19.8
1.5028 13.6 1.4946 16.8 1.4865 20.0
1.5023 13.8 1.4940 17.0 1.4860 20.2
1.5018 14.0 1.4935 17.2 1.4855 20.4
1.5012 14.2 1.4930 17.4 1.4850 20.6
1.5007 14.4 1.4925 17.6 1.4845 20.8
1.5002 14.6 1.4920 17.8 1.4840 21.0
1.4997 14.8 1.4915 18.0 1.4835 21.2
1.4992 15.0 1.4910 18.2 1.4830 21.4
1.4987 15.2 1.4905 18.4 1.4825 21.6
1.4982 15.4 1.4900 18.6 1.4820 21.8
1.4976 15.6 1.4895 18.8 1.4815 22.0
1.4971 15.8 1.4890 19.0
1.4966 16.0 1.4885 19.2
167
Annexure -II Color designation of honey samples: relationship of optical density and Pfund scale
Location of
Samples
Optical density Pfund Scale (mm) Color designation
Abdulhakeem 1.2789 50 to and 85 Light Amber
1.3871 50 to and 85 Light Amber
2.3188 85 to 114 Amber
1.0915 85 to 114 Amber
Bahawalpur 2.1925 85 to 114 Amber
5.0361 Over 114 Dark Amber
4.5127 Over 114 Dark Amber
2.1914 85 to 114 Amber
2.8635 85 to 114 Amber
1.5776 85 to 114 Amber
2.0393 85 to 114 Amber
Dunya Pur 2.0119 85 to 114 Amber
1.9582 85 to 114 Amber
2.3752 85 to 114 Amber
1.912 85 to 114 Amber
Faisalabad(Dwarf) 1.4637 85 to 114 Amber
1.6057 85 to 114 Amber
1.0391 50 to and 85 Light Amber
1.321 50 to and 85 Light Amber
1.5682 85 to 114 Amber
2.2627 85 to 114 Amber
Giant 2.3192 85 to 114 Amber
1.9192 85 to 114 Amber
Continued………..
168
Location of
Samples
Optical density Pfund Scale (mm) Color designation
1.684 85 to 114 Amber
1.9972 85 to 114 Amber
1.6959 85 to 114 Amber
Layyah 1.9001 85 to 114 Amber
1.8801 85 to 114 Amber
Multan 2.5903 85 to 114 Amber
1.8597 85 to 114 Amber
2.2689 85 to 114 Amber
2.1313 85 to 114 Amber
2.1759 85 to 114 Amber
1.999 85 to 114 Amber
2.0245 85 to 114 Amber
2.5934 85 to 114 Amber
2.0619 85 to 114 Amber
Shorkot 1.1808 50 to and 85 Light Amber
2.0341 85 to 114 Amber
2.2687 85 to 114 Amber
1.7241 85 to 114 Amber
Karror 2.6059 85 to 114 Amber
3.6431 Over 114 Dark Amber
3.0936 Over 114 Dark Amber
Beekeepers 1.0879 50 to and 85 Light Amber
0.7426 50 to and 85 Light Amber
0.7234 50 to and 85 Light Amber
1.1127 50 to and 85 Light Amber
0.0931 Darker than amber
Continued……..
169
Location of
Samples
Optical density Pfund Scale (mm) Color designation
1.0132 50 to and 85 Light Amber
Islamic Shahed 1.0079 50 to and 85 Light Amber
0.7854 50 to and 85 Light Amber
Marhaba honey 0.865 50 to and 85 Light Amber
China Honey 0.721 50 to and 85 Light Amber
Muqeet Honey 1.3113 50 to and 85 Light Amber
Langanese Honey 0.8658 50 to and 85 Light Amber
Swat honey 0.9762 50 to and 85 Light Amber
Young´s honey 0.5912 34 to 50 Extra Light Amber
Life Style 1.0799 50 to and 85 Light Amber
Cooperative 1.8518 85 to 114 Amber