1
STUDY OF RESIDUAL ANTIBIOTICS AND THEIR
METABOLITES IN HONEY
By
KHALIQ UR RAHMAN
Dissertation Submitted to the University of Peshawar in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY IN
CHEMISTRY
INSTITUTE OF CHEMICAL SCIENCES
UNIVERSITY OF PESHAWAR, PESHAWAR
PAKISTAN
(FABURARY, 2016)
2
STUDY OF RESIDUAL ANTIBIOTICS AND THEIR
METABOLITES IN HONEY
By
KHALIQ UR RAHMAN
DISSERTATION
SUBMITTED TO THE UNIVERSITY OF PESHAWAR IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
IN CHEMISTRY
INSTITUTE OF CHEMICAL SCIENCES
UNIVERSITY OF PESHAWAR, PAKISTAN
FABURARY, 2016
3
DECLARATIONS
This is to certify that this dissertation prepared by Mr. Khaliq
ur Rahman entitled “Study of Residual Antibiotics and Their
Metabolites in Honey” is accepted in the present form by the
Institute of Chemical Sciences, University of Peshawar as
fulfilling this part of the requirements for the degree
of
DOCTOR OF PHILOSOPHY IN CHEMISTRY
________________________ ________________________
SUPERVISOR CO-SUPERVISOR
Prof. Dr. Imdadullah Mohammadzai Dr. Arshad Hussain
Institute of Chemical Science, Senior Scientific Officer,
University of Peshawar, Peshawar, Food Technology Center,
Pakistan PCSIR Laboratories Complex
Peshawar, Pakistan
________________________ ________________________
EXTERNAL EXAMINER Prof. Dr. Yousaf Iqbal
Director,
Institute of Chemical Sciences,
University of Peshawar, Peshawar
Pakistan
4
THIS THESIS IS
DEDICATED TO
MY
PARENTS
(An Eternal Source of Guidance)
5
ACKNOWLEDGEMENT
In the name of Allah, the Most Gracious and the Most Merciful
Alhamdulillah, all praises to Allah for the strengths and His blessing in
completing this thesis. Special appreciation goes to my supervisor, Professor Dr.
Imdad Ullah Mohammadzai, for his supervision and constant support and knowledge
regarding this topic. Not forgotten, my appreciation to my co-supervisor, Dr. Arshad
Husain Senior Scientific Officer PCSIR lab complex Peshawar. For his invaluable
help of constructive comments and suggestions throughout the experimental and
thesis works have contributed to the success of this research.
I would like to express my appreciation to the Voice Chancellor University of
Peshawar, Dr Rasool Jan, Director Admission University of Peshawar Dr. Hizbullah,
Chairman of Pharmacy Department, Dr. Zafar Iqbal, Director Institute of Chemical
Science Dr Yousaf Iqbal, Prof. Dr. Imtiaz Ahmad, Dr. Waseem Hassan,
Dr.muhammad Imran khan and also for their support and help towards my
postgraduate affairs. My acknowledgement also goes to all the technicians and office
staffs of mycotoxin laboratory PCSIR as well Institute of chemical science for their
co-operations, especially Shafqatullah and Izhar Khan.
Sincere expressions of deep regards are due to all my teachers, friends and
staff members of this institute, for support during my study.
My deepest gratitude goes to my beloved parents; Mr. Hamid ur Rahman, my uncles,
also to my brothers, sisters and my cousins for their endless love and prayers. Last
but not least my uncle Dr Capt. Said Rahman for his encouragement. To those who
indirectly contributed in this research, your kindness means a lot to me. Thank you
very much. Khaliq ur Rahman
6
CONTENTS
S. NO. TITLE PAGE
1.
1.1.
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
SUMMARY
GENERAL INTRODUCTION
CHAPTER-1
INTRODUCTION
HONEY
I-IV
V-XIII
XIV-XVI
XVII-XXII
XXIII-XXIV
1
1
1.2. PRODUCTION AND CONSUMPTION OF HONEY 2
1.3. HISTORY OF HONEY 3
1.4. PHYSICAL PROPERTIES OF HONEY 4
1.5. CHEMICAL COMPOSITION OF HONEY 6
1.6. USES OF HONEY 8
7
1.7. CLASSIFICATION OF HONEY 10
1.7.1. CLASSIFICATION BASED ON PROCESSING 13
1.8.
1.9.
1.9.1.
1.9.2.
SPECIES OF HONEY BEES
DISEASES OF HONEY BEES
AMERICAN FOUL BROOD DISEASE
EUROPEAN FOUL BROOD DISEASE
17
22
22
23
1.10. ANTIBIOTICS 24
1.10.1. BRIEF HISTORY OF ANTIBIOTICS 25
1.10.2. CLASSIFICATION OF ANTIBIOTICS 29
1.10.3. ANTIBIOTICS ALLOWED IN BEEKEEPING 32
1.10.4. ANTIBIOTIC AS RESIDUES 34
1.10.5. METABOLITES OF ANTIBIOTICS 37
1.10.5.1. TYPES OF METABOLITES 37
1.10.5.2. ANTIBIOTIC METABOLITES IN HONEY 38
8
1.11. ANTIFUNGAL 41
1.11.1. ANTIFUNGAL ACTIVITY OF HONEY 42
1.11.2. ANTIBACTERIAL 42
1.11.3. ANTIBACTERIAL ACTIVITY OF HONEY 43
1.12. ANTIOXIDANTS 44
1.12.1. PHENOLIC COMPOUNDS 44
1.12.2. ANTIOXIDANT ACTIVITY OF HONEY 46
1.12.3. PROPERTIES OF PHENOLIC COMPOUNDS OF HONEY 47
1.13. PROXIMATE COMPOSITION OF HONEY 48
1.14. PHYTOCHEMICALS 49
1.14.1. PHYTOCHEMICALS COMPONENTS IN HONEY 51
1.15. CARBOHYDRATE 54
1.15.1. CARBOHYDRATES IN HONEY 55
1.16. CONTAMINATION IN HONEY 58
9
1.16.1. NON TOXIC HEAVY METALS 58
1.16.2. TOXIC HEAVY METALS 58
1.16.3. HEAVY METALS IN HONEY 60
1.17. MYCOTOXINS 61
1.17.1. AFLATOXINS 62
1.17.2. AFLATOXINS IN HONEY 64
1.18.
1.19.
AIMS AND OBJECTIVES
SUGGESTION FOR FURTHER WORK
65
65
CHAPTER-2
2.0. LITERATURE AND REVIEW 66
CHAPTER-3
3.0. MATERIALS AND METHODS
3.1. COLLECTION OF SAMPLES 80
3.2 CHEMICALS 89
10
3.3. PREPARATION OF REAGENTS 90
3.4. DETERMINATION OF ANTIBIOTICS 95
3.4.1. STANDARDS PREPARATION 95
3.4.2. EXTRACTION PROCEDURE FOR DETECTION ON TLC 95
3.4.3. EXTRACTION PROCEDURE FOR HPLC 95
3.4.4. TLC ANALYSIS OF ANTIBIOTICS 95
3.4.5. HPLC ANALYSIS OF ANTIBIOTICS 96
3.5. STANDARDS PREPARATION FOR SULFONAMIDE
ANTIBIOTICS
96
3.5.1. EXTRACTION PROCEDURE FOR HPLC 97
3.5.2.
3.6.
HPLC ANALYSIS OF SULFONAMIDE ANTIBIOTICS
CHLORAMPHENICOL
97
98
3.6.1. STANDARD PREPARATION 98
3.6.2.
3.6.3.
3.7.
EXTRACTION PROCEDURE FOR HPLC
HPLC ANALYSIS
DETERMINATION OF NITROFURAN AND THEIR
98
99
100
11
METABOLITES
3.7.1. PREPARATION OF STANDARD SOLUTIONS OF FZD & FTD 100
3.7.2.
3.7.3
3.7.4
3.7.5
3.7.6
3.7.7
EXTRACTION OF HONEY SAMPLE FOR FZD & FTD
CHROMATOGRAPHIC CONDITIONS
MATERIAL AND METHODS FOR AOZ AND AMOZ-D5
DERIVATIZATION OF AOZ & AMOZ
SAMPLE PREPARATION FOR DETECTION OF AOZ AND
AMOZ
CHROMATOGRAPHIC CONDITIONS
100
101
101
102
102
103
3.8. DETERMINATION OF ANTIOXIDANTS 103
3.8.1. EXTRACTION PROCEDURE 103
3.8.2. UV ANALYSIS 103
3.9. DETERMINATION OF PHENOLIC COMPOUNDS 104
3.9.1. STANDARD PREPARATION 104
3.9.2. HPLC ANALYSIS 104
12
3.10. DETERMINATION OF ANTIFUNGAL AND ANTIBACTERIAL
ACTIVITY
105
3.10.1.
3.10.2.
TEST ORGANISMS
PREPARATION OF HONEY SOLUTIONS
105
106
3.10.3. ANTIMICROBIAL ACTIVITY 106
3.10.4. MINIMUM INHIBITORY CONCENTRATION (MIC) 107
3.11.
3.11.1
3.11.2
3.11.3.
3.11.3.1.
3.11.3.2.
3.11.3.3.
3.11.3.4.
DETERMINATION OF PHYTOCHEMICALS
QUALITATIVE TESTS
QUANTITATIVE PROCEDURE
DETERMINATION OF CHEMICAL COMPOSITION
MOISTURE
ASH
PH AND TOTAL SOLUBLE SOLIDS
TOTAL ACIDITY
107
108
109
112
113
113
113
113
3.11.3.5. CRUDE FATS 114
3.11.3.6. CRUDE FIBER 114
13
3.11.3.7. REDUCING SUGAR 115
3.11.3.8. TOTAL SUGAR 116
3.11.3.9. NON-REDUCING SUGAR 116
3.11.3.10. MINERALS 117
3.11.3.11. H.M.F CONTENTS 117
3.12. DETERMINATION OF CARBOHYDRATES 117
3.12.1. STANDARD PREPARATION 117
3.12.2. EXTRACTION PROCEDURE 117
3.12.3. HPLC ANALYSIS 118
3.13. DETERMINATION OF HYDROXY METHYL FURFURAL 119
3.13.1. THERMAL TREATMENT 119
3.13.2. PROCEDURE OF HYDROXY METHYL FURFURAL 119
3.14. DETERMINATION OF CONTAMINANTS 119
3.14.1. AFLATOXINS EXTRACTION PROCEDURE 119
14
3.14.2. TLC ANALYSIS 120
3.14.3.
3.14.3.1.
3.14.3.2.
3.14.3.3.
DETERMINATION OF HEAVY METALS
EXTRACTION PROCEDURE
ATOMIC ABSORPTION SPECTROPHOTOMETER ANALYSIS
STATISTICAL ANALYSIS
120
120
121
122
CHAPTER-4
4.0. RESULTS AND DISCUSSIONS
4.1. ANTIBIOTICS 123
4.1.1. SULFONAMIDE 129
4.1.2. CHLORAMPHENICOL 132
4.1.3. NITROFURANS 133
4.2. ANTIOXIDANTS 147
4.3. PHENOLIC ACIDS 153
4.4. ANTIFUNGAL ANTIBACTERIAL 159
4.5. PHYTOCHEMICALS 178
15
4.6 CHEMICAL COMPOSITION 184
4.7. CARBOHYDRATES 199
4.8. HYDROXY METHYL FURFURAL 209
4.9. CONTAMINANTS 223
CONCLUSIONS 232
REFERENCES 235
LIST OF PUBLICATIONS 261
16
17
LIST OF TABLES
TABLES
NO.
TITLE PAGE
1.0 LIST OF APPROVED PRODUCTS IN APICULTURE 33
3.1 INSTRUMENTAL CONDITIONS FOR THE MAINTAIN OF
EACH ELEMENT FOR
ATOMIC ABSORPTION SPECTROPHOTOMETER
121
4.1 DETECTION OF ANTIBIOTIC RESIDUES IN HONEY
SAMPLES
125
4.2 CONCENTRATION OF ANTIBIOTICS RESIDUES IN
BRANDED HONEY (MG/KG)
125
4.3 CONCENTRATION OF ANTIBIOTICS RESIDUES IN
UNBRANDED
HONEY (MG/KG)
126
4.4 CONCENTRATION OF ANTIBIOTICS RESIDUES IN
NATURAL HONEY (MG/KG)
126
18
4.5 CONCENTRATION OF SULFONAMIDE ANTIBIOTIC IN
BRANDED HONEY SAMPLES
130
4.6 CONCENTRATION OF SULFONAMIDE ANTIBIOTIC IN
UNBRANDED HONEY SAMPLES
131
4.7 CONCENTRATION OF SULFONAMIDE ANTIBIOTIC IN
NATURAL HONEY SAMPLES
131
4.8 CONCENTRATION OF CHLORAMPHENICOL
ANTIBIOTIC RESIDUES IN BRANDED HONEY SAMPLES
132
4.9 CONCENTRATION OF CHLORAMPHENICOL
ANTIBIOTIC RESIDUES IN UNBRANDED HONEY
SAMPLES
133
4.10 CONCENTRATION OF CHLORAMPHENICOL
ANTIBIOTIC RESIDUES IN NATURAL HONEY SAMPLES
133
4.11 CONCENTRATION OF NITROFURAN AND THEIR
METABOLITES IN BRANDED HONEY SAMPLES
146
4.12 CONCENTRATION OF NITROFURAN AND THEIR
METABOLITES IN UNBRANDED HONEY SAMPLES
147
19
4.13 CONCENTRATION OF NITROFURAN AND THEIR
METABOLITES IN NATURAL HONEY SAMPLES
147
4.14 DPPH RADICAL SCAVENGING ACTIVITY OF
BRANDED HONEY SAMPLES
151
4.15 DPPH RADICAL SCAVENGING ACTIVITY OF
UNBRANDED HONEY SAMPLES
152
4.16 DPPH RADICAL SCAVENGING ACTIVITY OF
NATURAL COMB HONEY SAMPLES
152
4.17 DPPH RADICAL SCAVENGING ACTIVITY OF
BRANDED HONEY
(EC50 IN µG/G)
152
4.18 DPPH RADICAL SCAVENGING ACTIVITY OF
UNBRANDED HONEY
(EC50 IN µG/G)
153
4.19 DPPH RADICAL SCAVENGING ACTIVITY OF
NATURAL COMB HONEY
153
20
(EC50 IN µG/G)
4.20 CONCENTRATION OF PHENOLIC ACIDS IN
BRANDED HONEY (MG/100G)
156
4.21 CONCENTRATION OF PHENOLIC ACIDS IN UNBRANDED
HONEY
(MG/100G)
156
4.22 CONCENTRATION OF PHENOLIC ACIDS IN NATURAL
HONEY (MG/100G)
156
4.23 ANTIFUNGAL ACTIVITY OF BRANDED HONEY
AGAINST CANDIDA. ALBICANSAND
ASPERGILLUSNIGER
163
4.24 ANTIFUNGAL ACTIVITY OF UNBRANDED HONEY
AGAINST CANDIDA. ALBICANS AND
ASPERGILLUSNIGER
163
4.25 ANTIFUNGAL ACTIVITY OF NATURAL HONEY
AGAINST C. ALBICANS AND ASPERGILLUS NIGER
164
4.26 MIC OF BRANDED HONEY AGAINST C. ALBICANS
21
AND ASPERGILLUS NIGER. % (V/V) 164
4.27 MIC OF UNBRANDED HONEY AGAINST C.
ALBICANSAND ASPERGILLUSNIGER. % (V/V)
164
4.28 MIC OF NATURAL COMB HONEY AGAINST C.
ALBICANSAND ASPERGILLUSNIGER. % (V/V)
165
4.29 ANTIBACTERIAL ACTIVITY OF BRANDED HONEY
AGAINST E. COLI AND BACILLUS CEREUS
165
4.30 ANTIBACTERIAL ACTIVITY OF UNBRANDED HONEY
AGAINST E. COLI AND BACILLUS CEREUS
165
4.31 ANTIBACTERIAL ACTIVITY OF NATURAL COMB
HONEY AGAINST E. COLI AND BACILLUS CEREUS
166
4.32 MIC OF BRANDED HONEY AGAINST E.COLI AND
BACILLUS CEREUS (V/V %)
166
4.33 MIC OF UNBRANDED HONEY AGAINST E. COLI
AND BACILLUS CEREUS (V/V %)
166
4.34 MIC OF NATURAL COMB HONEY AGAINST E. COLI AND
BACILLUSCEREUS
167
22
4.35 QUALITATIVE TEST FOR PHYTOCHEMICALS IN BRANDED
HONEY SAMPLES
179
4.36 QUALITATIVE TEST FOR PHYTOCHEMICALS IN
UNBRANDED HONEY SAMPLES
180
4.37 QUALITATIVE TEST FOR PHYTOCHEMICALS IN
NATURAL COMB HONEY SAMPLES
180
4.38 QUANTITATIVE TEST FOR PHYTOCHEMICALS IN
BRANDED HONEY SAMPLES
182
4.39 QUANTITATIVE TEST FOR PHYTOCHEMICALS IN
UNBRANDED HONEY SAMPLES
182
4.40 QUANTITATIVE TEST FOR PHYTOCHEMICALS IN
NATURAL COMB HONEY SAMPLES
183
4.41 CHEMICAL COMPOSITION OF BRANDED HONEY
SAMPLES
186
4.42 CHEMICAL COMPOSITION OF UNBRANDED HONEY
SAMPLES
187
23
4.43 CHEMICAL COMPOSITION OF NATURAL COMB
HONEY SAMPLES
189
4.44 CARBOHYDRATES CONCENTRATION IN BRANDED
FARMS HONEY (G/100G)
202
4.45 CARBOHYDRATES CONCENTRATION IN UNBRANDED
HONEY (G/100G)
203
4.46 CARBOHYDRATES CONCENTRATION IN NATURAL
COMB HONEY (G/100G)
205
4.47 EFFECT OF TEMPERATURE ON H.M.F
CONCENTRATION IN FARMS HONEY
214
4.48 EFFECT OF TEMPERATURE ON H.M.F
CONCENTRATION IN NATURAL HONEY
215
4.49 EFFECT OF FLAME HEATING ON H.M.F
CONCENTRATION IN FARMS HONEY
216
4.50 EFFECT OF FLAME HEATING ON H.M.F
CONCENTRATION IN NATURAL HONEY
217
24
4.51 HEAVY METALS CONCENTRATION IN BRANDED
HONEY (µG/KG)
224
4.52 HEAVY METALS CONCENTRATION IN UNBRANDED
HONEY (µG/KG)
224
4.53 HEAVY METALS CONCENTRATION IN NATURAL
COMB HONEY (µG/KG)
225
4.54 MYCOTOXIN CONCENTRATION IN BRANDED
HONEY (µG/KG)
225
4.55 MYCOTOXIN CONCENTRATION IN UNBRANDED
HONEY (µG/KG)
226
4.56 MYCOTOXIN CONCENTRATION IN NATURAL COMB
HONEY (µG/KG)
226
LIST OF FIGURES
25
FIGURE
NO
TITLE PAGE
1.1A HONEY 1
1.2A HONEY PRODUCTION BY COUNTRY 2
1.2B WORLD PER YEAR HONEY CONSUMPTION 2
1.3A CHEMICAL COMPOSITION OF HONEY 7
1.4A TRADITIONAL AND MODERN USES OF HONEY 9
1.5A CLASSIFICATION OF HONEY 11
1.5B CLASSIFICATION OF HONEY ON THE BASIS OF
SOURCES AND PROCESSING
12
1.5C HYDROXY METHYL FURFURAL STRUCTURE 13
1.5D HONEY PROCESSING PLANT 14
1.5E PROCESSING OF HONEY EXTRACTION 15
1.5F TOOLS OF PROCESSING AND EXTRACTION OF 16
26
HONEY
1.6A STINGLESS BEE’S 18
1.6B BUMBLE BEE’S 19
1.6C APISMELLIFERA 20
1.6D APISFLOREA 21
1.6E APISDORSATA 21
1.6F APISCERANA 22
1.7A AMERICAN FOULBROOD DISEASES 23
1.7B EUROPEAN FOULBROOD DISEASES 24
1.8A DISCOVERY OF DIFFERENT ANTIBIOTICS FROM 1940
TO 2000
25
1.8B BRIEF HISTORY OF ANTIBIOTICS
26
1.8C PENICILLIN STRUCTURE 26
1.8D STREPTOMYCIN STRUCTURE 27
27
1.8E CHLORAMPHENICOL STRUCTURE 27
1.8F OXYTETRACYCLINE STRUCTURE 28
1.8G CLASSIFICATION OF ANTIBIOTICS , USES AND SIDE
EFFECTS ON HUMANS
31
1.8H GENTAMYCIN STRUCTURE 32
1.8I SULFONAMIDES STRUCTURES 36
1.8J NITROFURANS ANTIBIOTICS AND THEIR
METABOLITES STRUCTURES
40
1.9A PHENOLIC COMPOUND STRUCTURES 45
1.9B PHENOLIC ACIDS STRUCTURES 47
1.10A PHYTOCHEMICALS COMPOSITION CHART 50
1.10B PHYTOCHEMICALS STRUCTURES 53
1.11A CARBOHYDRATES CHART 54
1.11B CARBOHYDRATES STRUCTURES 55
28
1.11C CARBOHYDRATES STRUCTURES 56
1.12A TOXIC HEAVY METALS 59
1.12B AFLATOXINS B1, B2, G1 AND G2 STRUCTURES 63
3.1A HONEY BEE BOXES IN FARM 80
3.1B HONEY BEE BOXES IN FARM 81
3.1C PALOSA (ACACIA MODESTA) 81
3.1D SPERKAY (TRACHYSPERMUM) 81
3.1E BEKERR (JUSTICIA) 82
3.1F GRANDA (CARISSA OPACA) 82
3.1G BEERA (ZIZIPHUS) 82
3.2A BRANDED HONEY SAMPLES 83
3.2B BRANDED HONEY SAMPLES 83
3.3A UNBRANDED HONEY SAMPLES 85
29
3.4A NATURAL COMB HONEY SAMPLES 87
3.4B SAMPLE COLLECTION FROM COMB 87
3.5A ANTIBIOTICS USED FOR HONEY BEES 88
3.6A TEST ORGANISMS PLATES 105
3.6B TEST ORGANISMS IMAGES 106
4.1A HPLC CHROMATOGRAM OF HONEY SAMPLE:
5.63 OXYTETRACYCLINE RESIDUE, OTHER
PEAKS AT 3.45, 4.63, AND 14.92 NOT IDENTIFIED
127
4.1B HPLC CHROMATOGRAM OF HONEY SAMPLE: 2.60
PENICILLIN RESIDUES, OTHER PEAKS AT 4.98, 6.78,
12.34, 14.45 AND 16.23 NOT IDENTIFIED
128
4.1C HPLC CHROMATOGRAM OF HONEY SAMPLE:
10.96 STREPTOMYCIN RESIDUE, OTHER PEAKS
AT 4.61, 7.23, 8.02, AND 13.49 NOT IDENTIFIED
128
4.1D CONCENTRATION OF ANTIBIOTIC RESIDUES IN
BRANDED HONEY SAMPLES
128
4.1E CONCENTRATION OF ANTIBIOTIC RESIDUES IN
UNBRANDED HONEY SAMPLES
129
30
4.2A HPLC CHROMATOGRAM OF SULFONAMIDES
STANDARD, 13.20 SULFACETAMIDE (SCA), 14.10
SULFAMETHAZINE (SMT) AND 15.05 SULFATHIAZOLE
(STZ) WERE IDENTIFIED
130
4.2B
4.2C
4.2D
4.2E
4.2F
4.2G
LCMS-MS CHROMATOGRAM OF NITROFURAN
METABOLITES STANDARD, 3.90 AOZ=3-AMINO-2-
OXAZOLIDINONE; 4.16 AMOZ = 3-AMINO-5-
MORPHOLINO-METHYL-1, 3-OXA- ZOLIDINONE;
WERE IDENTIFIED.
LCMS-MS CHROMATOGRAM OF FURAZOLIDONE
AOZ=3-AMINO-2-OXAZOLIDINONE
LCMS-MS CHROMATOGRAM OF FURALTADONE
AMOZ = 3-AMINO-5-MORPHOLINO-METHYL-1, 3-
OXA- ZOLIDINONE
STANDERD COLIBRATION; LIMIT OF DETECTION (LOD)
AND LIMIT OF QUANTIFICATION (LOQ) OF
FURAZOLIDONE
STANDERD COLIBRATION; LIMIT OF DETECTION (LOD)
AND LIMIT OF QUANTIFICATION (LOQ) OF
FURALTADONE
LCMS-MS CHROMATOGRAM OF HONEY SAMPLE
135
136
137
138
31
4.2H
4.2I
4.2J
4.2K
4.2L
4.2M
LCMS-MS CHROMATOGRAM OF FURAZOLIDONE
AOZ=3-AMINO-2-OXAZOLIDINONE
LCMS-MS CHROMATOGRAM OF FURAZOLIDONE
AOZ=3-AMINO-2-OXAZOLIDINONE
LCMS-MS CHROMATOGRAM OF FURALTADONE
AMOZ = 3-AMINO-5-MORPHOLINO-METHYL-1, 3-
OXA- ZOLIDINONE
LCMS-MS CHROMATOGRAM OF FURALTADONE
AMOZ = 3-AMINO-5-MORPHOLINO-METHYL-1, 3-
OXA- ZOLIDINONE
LCMS-MS CHROMATOGRAM OF FURALTADONE
AMOZ = 3-AMINO-5-MORPHOLINO-METHYL-1, 3-
OXA- ZOLIDINONE IN HONEY
LCMS-MS CHROMATOGRAM OF NITROFURAN
METABOLITES STANDARD,13.0 NITROFURANTOIN
AHD= 1-AMINOHYDANTOIN; 13.8
NITROFURAZONE SEM = SEMICARBAZIDE; WERE
IDENTIFIED.
139
140
141
142
143
144
32
145
146
4.3A ANTIOXIDANT ACTIVITY OF BRANDED HONEY
SAMPLES
148
4.3B ANTIOXIDANT ACTIVITY OF UNBRANDED HONEY
SAMPLES
149
4.3C
4.3D
ANTIOXIDANT ACTIVITY OF NATURAL COMB HONEY
SAMPLES
1, 1-DIPHENYL-2-PICRYL HYDROXYL STRUCTURE
149
150
4.4A HPLC CHROMATOGRAM OF PHENOLIC ACIDS
STANDARD, 2.05 GALLIC ACID, 6.45 CHLOROGINIC
ACID, 10.15 SYRINGICACID, 12.05 BENZOIC ACID,
154
33
21.52 VANILLIC ACID WERE IDENTIFIED
4.4B CONCENTRATION OF PHENOLIC ACID IN
BRANDED HONEY SAMPLES
158
4.4C CONCENTRATION OF PHENOLIC ACID IN
UNBRANDED HONEY SAMPLES
158
4.4D CONCENTRATION OF PHENOLIC ACID IN
NATURAL COMB HONEY SAMPLES
159
4.5A ANTIFUNGAL ACTIVITY OF BRANDED HONEY
SAMPLES AGAINST ASPERGILLUSNIGER
167
4.5B ANTIFUNGAL ACTIVITY OF UNBRANDED HONEY
SAMPLES AGAINST ASPERGILLUSNIGER
167
4.5C ANTIFUNGAL ACTIVITY OF NATURAL COMB HONEY
SAMPLES AGAINST ASPERGILLUSNIGER
168
4.5D MINIMUM INHIBITORY CONCENTRATION OF
BRANDED HONEY SAMPLES AGAINST CANDIDA
ALBICANS
169
4.5E MINIMUM INHIBITORY CONCENTRATION OF 169
34
BRANDED HONEY SAMPLES AGAINST
ASPERGILLUSNIGER
4.5F MINIMUM INHIBITORY CONCENTRATION OF
UNBRANDED HONEY SAMPLES AGAINST CANDIDA
ALBICANS
170
4.5G MINIMUM INHIBITORY CONCENTRATION OF
UNBRANDED HONEY SAMPLES AGAINST
ASPERGILLUSNIGER
170
4.5H
4.5I
MINIMUM INHIBITORY CONCENTRATION OF
NATURAL COMB HONEY SAMPLES AGAINST
CANDIDA ALBICANS
MINIMUM INHIBITORY CONCENTRATION OF
NATURAL COMB HONEY SAMPLES AGAINST
CANDIDA ALBICANS
171
171
4.5J ANTIBACTERIAL ACTIVITY OF BRANDED HONEY
SAMPLES AGAINST E.COLI
172
4.5K ANTIBACTERIAL ACTIVITY OF BRANDED HONEY
SAMPLES AGAINST BACILLUS CEREUS
172
35
4.5L ANTIBACTERIAL ACTIVITY OF UNBRANDED HONEY
SAMPLES AGAINST E.COLI
173
4.5M ANTIBACTERIAL ACTIVITY OF UNBRANDED HONEY
SAMPLES AGAINST BACILLUS CEREUS
173
4.5N ANTIBACTERIAL ACTIVITY OF NATURAL COMB
HONEY SAMPLES AGAINST E.COLI
174
4.5O ANTIBACTERIAL ACTIVITY OF NATURAL COMB
HONEY SAMPLES AGAINST BACILLUS CEREUS
174
4.5P MINIMUM INHIBITORY CONCENTRATION OF
BRANDED HONEY SAMPLES AGAINST E.COLI
175
4.5Q MINIMUM INHIBITORY CONCENTRATION OF
BRANDED HONEY SAMPLES AGAINST BACILLUS
CEREUS
175
4.5R MINIMUM INHIBITORY CONCENTRATION OF
UNBRANDED HONEY SAMPLES AGAINST E.COLI
176
4.5S MINIMUM INHIBITORY CONCENTRATION OF
UNBRANDED HONEY SAMPLES AGAINST BACILLUS
176
36
CEREUS
4.5T
4.5U
MINIMUM INHIBITORY CONCENTRATION OF
NATURAL COMB HONEY SAMPLES AGAINST E.COLI
MINIMUM INHIBITORY CONCENTRATION OF
NATURAL COMB HONEY SAMPLES AGAINST
BACILLUS CEREUS
177
177
4.6A CONCENTRATION OF PHYTOCHEMICALS IN
BRANDED HONEY SAMPLES
183
4.6B CONCENTRATION OF PHYTOCHEMICALS IN
UNBRANDED HONEY SAMPLES
184
4.6C CONCENTRATION OF PHYTOCHEMICALS IN
NATURAL COMB HONEY SAMPLES
184
4.6D (I) CHEMICAL COMPOSITION OF BRANDED HONEY
SAMPLES
190
4.6D (II) CHEMICAL COMPOSITION OF BRANDED HONEY
SAMPLES
190
4.6E (I) CHEMICAL COMPOSITION OF UNBRANDED HONEY 191
37
SAMPLES
4.6E (II) CHEMICAL COMPOSITION OF UNBRANDED HONEY
SAMPLES
191
4.6F (I) CHEMICAL COMPOSITION OF NATURAL COMB
HONEY SAMPLES
192
4.6F (II) CHEMICAL COMPOSITION OF NATURAL COMB
HONEY SAMPLES
192
4.6G PH CONCENTRATION OF BRANDED HONEY SAMPLES 193
4.6H PH CONCENTRATION OF UNBRANDED HONEY
SAMPLES
193
4.6I PH CONCENTRATION OF NATURAL COMB HONEY
SAMPLES
194
4.6J ACIDITY CONCENTRATION OF BRANDED HONEY
SAMPLES
194
4.6K ACIDITY CONCENTRATION OF UNBRANDED HONEY
SAMPLES
195
38
4.6L ACIDITY CONCENTRATION OF NATURAL COMB
HONEY SAMPLES
195
4.6M ELECTRICAL CONDUCTIVITY CONCENTRATION OF
BRANDED HONEY SAMPLES
196
4.6N ELECTRICAL CONDUCTIVITY CONCENTRATION OF
UNBRANDED HONEY SAMPLES
196
4.6O ELECTRICAL CONDUCTIVITY CONCENTRATION OF
NATURAL HONEY SAMPLES
197
4.6P HYDROXY METHYL FURFURAL CONCENTRATION IN
BRANDED HONEY
197
4.6Q HYDROXY METHYL FURFURAL CONCENTRATION IN
UNBRANDED HONEY
198
4.6R HYDROXY METHYL FURFURAL CONCENTRATION IN
NATURAL COMB HONEY
198
4.7A HPLC CHROMATOGRAM OF CARBOHYDRATES
STANDARD: 5.03 PMP, 8.02 MANOSE, 11.03 RIBOSE,
13.45 LACTOSE, 17.52 MALTOSE, 23.01 SUCROSE,
27.50 GLUCOSE, 31.50 XYLOSE, 32.08 GLACTOSE,
36.52 ARABINOSE AND 39.02 FOR FRUCTOSE WERE
199
39
IDENTIFIED.
4.7B (I) CONCENTRATION OF MAJOR CARBOHYDRATES IN
BRANDED HONEY SAMPLES
206
4.7B (II) CONCENTRATION OF MINOR CARBOHYDRATES IN
BRANDED HONEY SAMPLES
207
4.7C (I) CONCENTRATION OF MAJOR CARBOHYDRATES IN
UNBRANDED HONEY SAMPLES
207
4.7C (II) CONCENTRATION OF MINOR CARBOHYDRATES IN
UNBRANDED HONEY SAMPLES
208
4.7D (I) CONCENTRATION OF MAJOR CARBOHYDRATES IN
NATURAL HONEY SAMPLES
208
4.7D (II) CONCENTRATION OF MINOR CARBOHYDRATES IN
NATURAL HONEY SAMPLES
209
4.8A INITIAL CONCENTRATION OF H.M.F IN HONEY
SAMPLES
218
4.8B INITIAL CONCENTRATION OF HYDROXY METHYL
FURFURAL IN NATURAL
219
40
HONEY SAMPLES
4.8C EFFECT OF TEMPRATURE (35°C) ON HYDROXY
METHYL FURFURAL CONCENTRATION IN FARM
HONEY SAMPLES
219
4.8D EFFECT OF TEMPRATURE (50°C) ON HYDROXY
METHYL FURFURAL CONCENTRATION IN FARM
HONEY SAMPLES
220
4.8E EFFECT OF TEMPRATURE (70°C) ON HYDROXY
METHYL FURFURAL CONCENTRATION IN FARM
HONEY SAMPLES
220
4.8F
EFFECT OF TEMPRATURE (35°C) ON HYDROXY
METHYL FURFURAL CONCENTRATION IN NATURAL
HONEY SAMPLES
221
4.8G EFFECT OF TEMPERATURE (50°C) ON H.M.F
CONCENTRATION IN NATURAL HONEY SAMPLES
221
4.8H EFFECT OF TEMPRATURE (70°C) ON HYDROXY
METHYL FURFURAL CONCENTRATION IN NATURAL
HONEY SAMPLES
222
41
4.8I EFFECT OF FLAME HEATING ON HYDROXY METHYL
FURFURAL CONCENTRATION IN FARM HONEY
SAMPLES
222
4.8J EFFECT OF FLAME HEATING ON HYDROXY METHYL
FURFURAL CONCENTRATION IN NATURAL COMB
HONEY SAMPLES
223
4.9A CONCENTRATION OF HEAVY METALS IN BRANDED
HONEY SAMPLES
230
4.9B CONCENTRATION OF HEAVY METALS IN
UNBRANDED HONEY SAMPLES
230
4.9C CONCENTRATION OF HEAVY METALS IN NATURAL
COMB HONEY SAMPLES
231
4.9D CONCENTRATION OF MYCOTOXIN IN BRANDED
HONEY SAMPLES
231
4.9E CONCENTRATION OF MYCOTOXIN IN UNBRANDED
HONEY SAMPLES
232
42
43
LIST OF ABBREVIATIONS
ABBREVIATION MEANINGS
AB ABSORBANCE OF BLANK
AS ABSORBANCE OF TEST SAMPLE
ACN ACETONITRILE
ADIS ACCEPTABLE DAILY INTAKES
AF B1, B2, G1, G2 AFLATOXINS, BLUE, GREEN
AMP AMPICILLIN
AMOZ 3-AMINO-5-MORPHOLINOMETHYL-2-
OXAZOLIDINONE
AOAC ASSOCIATION OF OFFICIAL ANALYTICAL
CHEMISTS
AHD 1-AMINOHYDANTOIN HYDROCHLORIDE,
AOZ 3-AMINO-2-OXAZOLIDINONE,
44
ANOVA ANALYSIS OF VARIANCE
ATSDR AGENCY FOR TOXIC SUBSTANCES AND
DISEASE REGISTRY
ATCC AMERICAN TYPE CULTURE COLLECTION
AHD 1-AMINOHYDANTOIN
APA AMERICAN PSYCHOLOGICAL
ASSOCIATION
CFU/ML COLONY FORMING UNITS PER MILLILITER
C. ALBICANS CANDIDA. ALBICANS
DMSO DI-METHYL SULFHOXIDE
°C DEGREE CENTIGRADE
DPPH 1,1-DIPHENYL-2-PICRYL HYDROXYL
DNA DEOXYRIBONUCLEIC ACID
E.COLI ESCHERICHIA COLI
45
ERY ERYTHROMYCIN
ENR ENROFLOXACIN
EC EFFECTIVE CONCENTRATION
ESI ELECTRO SPRAY IONIZATION
EU EUROPEAN UNION
FAO FOOD AGRICULTURE ORGANIZATION
FZD FURAZOLIDONE
FTD FURALTADONE
HMF HYDROXY METHYL FURFURAL
KM/H KILOMETER PER HOUR
LC-MS LIQUID CHROMATOGRAPHY/MASS
SPECTROMETRY
HPLC HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
46
MIC MINIMUM INHIBITORY CONCENTRATION
MG/ML MILLIGRAM PER MILLILITER
MG/G MILLIGRAM PER GRAM
MRLS MAXIMUM RESIDUE LIMITS
MM MILLIMETER
µG/ML MICROGRAM PER MILLILITER
µG/D, MICROGRAM PER DAY
MIC MINIMUM INHIBITORY CONCENTRATION
ND: NOT DETECTED
NFT NITROFURANTOIN
N NORMALITY
NFZ NITROFURAZONE
OTC OXYTETRACYCLINE
47
P. AERUGINOSA PSEUDOMONAS AERUGINOSA
PH POWER OF HYDROGEN ION CONCENTRATION
PDA DEXTROSE AGAR PLATE
PMP 1-PHENYL- 3-METHYL-5-PYRAZOLONE
KG -1 PER KILOGRAM
RF RETENTION FACTOR
S POSITIVE SAMPLES
SC SEMICARBAZIDE ,2-NITROBENZALDEHYDE,
SW SAMPLE WEIGHT
SV SAMPLE VOLUME
TLC THIN LAYER CHROMATOGRAPHY
TCS TOTAL COUNTED SAMPLES
UV-VIS ULTRAVIOLET-VISIBLE
48
WHO WORLD HEALTH ORGANIZATION
W WEIGHT
ZDI ZONE DIAMETER INHIBITION
49
SUMMARY
In this work evaluation of honey for the detection and quantification of antibiotic
residues such as oxytetracycline, streptomycin, gentamycin, penicillin, sulfonamide,
chloramphenicol, nitrofuran and their metabolites were performed. The metabolic
extract of branded, unbranded and natural honey samples were evaluated for their
scavenging activity of 1,1-diphenyl-2-picryl hydroxyl (DPPH) free radical by using
different concentrations (100, 200, 300, 500 and 600 µg/ml) of honey samples. The
phenolic acids, antimicrobial activities, nutritional significance and phytochemicals in
branded, unbranded and natural combs honey were also evaluated. This study was
also focused to evaluate the carbohydrates and Hydroxy Methyl Furfural (HMF)
content as well as the contamination level of aflatoxins (B1, B2, G1, and G2) and
heavy metals (cadmium, manganese, lead, mercury, nickel and cobalt) in branded,
unbranded and natural honey.
A total 100 samples of honey were collected from market of Khyber
Pakhtunkhwa, Pakistan and categorized as branded, unbranded and natural for
comparative study. The branded, unbranded and natural comb honey samples under
study were Marhaba, Qarshi, Versatile, Al-hayat, Young’s, Pak-salman, Langnese,
Big bees honey, Small bees honey, Beera, Palosa, Sperkay, Bekerr and Granda.
The detection of antibiotics such as tetracycline, streptomycin, gentamycin,
and penicillin residues was carried out by thin layer chromatography (TLC) method
while the positive samples were quantified by an optimized HPLC-UV method. The
sulfonamide residues such as sulfamethazine, sulfacetamide, sulfathiazole and
chloramphenicol residues were analyzed by HPLC. Nitrofuran and their metabolites
were determined by LC-MS-MS Technique.
50
The antioxidant activity of the extracts were determined against 1, 1 -diphenyl-2-
picryl hydroxyl (DPPH) by spectrophotometer. Five phenolic acids (chloroganic,
gallic, vanallic, benzoic and syringic) were identified and quantified by HPLC
technique, using UV-VIS Detector.
Antimicrobial activities evaluated by Disc diffusion (Mueller-Hinton Agar).
Different dilutions of honey were made against Candida albicans (ATCC Code
90028), Aspergillusniger (PCSIR 001), Escherichia coli (ATCC Code 35218) and
Bacillus cereus (ATCC Code 11778) for Minimum Inhibitory Concentration (MIC).
The chemical composition included such as; total ash, pH, moisture, total
acidity, electrical conductivity and total sugars were analyzed by standard methods of
AOAC. The photochemical such as tannins, phlobatanins, flavonoids, terpenoids,
glycosides, saponins, alkaloids and fluorides of branded, unbranded and natural comb
honey samples were carried out by UV-Spectrophotometer.
Carbohydrates such as alpha lactose, maltose, beta d-glucose, xylose, fructose,
ribose, mannose, arabinose, glactose and sucrose were identified and quantified
HPLC using UV-VIS Detector. The Hydroxy methyl furfural (HMF) content was
determined by Winkler’s method. The effects of flame and oven heating on HMF
content of honey were also checked. The samples were kept at different temperature
for different time period. The hydroxy methyl furfural (HMF) contents were
determined using spectrophotometer. Heavy metals concentration was detected using
atomic absorption spectrophotometer method.
About 12.5% of branded sample and 19.96% unbranded samples were found
positive while in all natural honey samples were found negative. Oxytetracycline
residue was found maximum in unbranded sample, while gentamycin was not
detected in any tested sample by TLC method. The quantification by HPLC the total
51
streptomycin residue was determined 16.31μg/g in five positive unbranded sample
while this residue was found to be minimum (3.6 µg/ml) in unbranded sample. The
sulfonamides, chloramphenicol and nitrofuran residues and their metabolites were not
detected in any sample.
In case of branded honey, Al-hayat honey showed maximum antioxidant
activity (81.26±1.44) at the concentration 600 µg/ml among all honey samples,
whereas the lowest activity (20.22±1.19) was observed at the concentration 100 µg/ml
in Marhaba honey. Unbranded, Small bees honey showed maximum antioxidant
activity (84.33±1.23) at the concentration 600 µg/ml, whereas the lowest activity
(24.12±1.17) was observed at the concentration 100 µg/ml in Beera honey. In natural
honey Big bees honey showed maximum antioxidant activity (85.22±1.23) at the
concentration 600 µg/ml, whereas the lowest activity (10.11±1.34) was observed at
the concentration 100 µg/ml in Beera honey. As the concentration of these
compounds increased the percent scavenging activity also increased.
The phenolic acids contents were found higher in all natural honey samples as
compared to branded and unbranded honey. Among the natural honey samples, the
maximum concentration (4.26mg/100g) of phenolic Acids was found in Palosa honey
while minimum (1.93mg/100g) in Bekerr honey sample. Similarly the maximum
concentration (2.78mg/100g) was found in Langnese honey, while minimum
(0.71mg/100g) in Versatile honey sample. In unbranded honey maximum
concentration (2.46mg/100g) was found in Beera honey, while minimum
(0.62mg/100g) in Palosa honey sample.
Maximum antifungal activities 14% have been shown by natural (Big bees honey),
while minimum activity 1% by branded (Young’s) and unbranded (Bekerr) honey
against Aspergillusniger. Maximum MICs 88% and 93% were observed in branded
52
(Marhaba) and unbranded (Big bee’s honey), while minimum MIC 35% were
observed in branded (Langnese) honey against Candida albicans. Maximum
Antibacterial activities 34mm and 35mm also been observed in branded (Qarshi) and
unbranded (Big bees honey) respectively, while minimumactivity 1mm and 2mm
found inbranded (Langnese) and unbranded (Palosa) against E. coli. Maximum MICs
90% and 93% observed in branded (Marhaba) and unbranded (Big bees honey)
against Bacillus cereus, while minimum MICs 3% and 4% in branded (Langnese
honey) and unbranded (Granda honey) respectively.
In branded honey, a maximum chemical composition (92.67%) was observed
in versatile honey, minimum (65.27%) in Qarshi honey. In unbranded honey,
maximum chemical composition (99.04%) was observed in Palosa honey, minimum
(74.03%) in Small bee’s honey. In natural comb honey, maximum chemical
composition (93.05%) was observed in Beera honey, minimum (78.34%) in Small
bee’s honey.
Among the branded honey sample, maximum concentration (78.00g/100g)
was found in Langnese honey, while minimum (54.25g/100g) in Al-hayat honey
sample. In unbranded honey maximum concentration (76.10g/100g) was found in
Beera honey, while minimum (54.84g/100g) in Sperkay honey sample.
Similarly in natural honey’s sample, maximum concentration (77.22g/100g) of
carbohydrates was found in Beera honey while minimum (70.18g/100g) in Sperkay
honey sample.
The H.M.F contents increased in all farm honey’s samples ranged from 100-
159% kept for 60 minutes at 70oC oven. The H.M.F contents increased in all natural
honey’s samples ranged from 124-144% when kept for 60 minutes at 70 0C in oven.
The H.M.F contents increased in all farm honey’s samples ranged from 407-593% for
53
12 minutes by flam heating. The HMF contents increased in all natural honey’s
samples ranged from 519-673% kept for 12 minutes by flame heating.
Higher concentration (µg / kg) of heavy metals was found in branded honey as
compared to unbranded and natural honey. As in Marhaba, Ni concentration
(0.49±0.03) found maximum while Co (0.15±0.02) was lowest. Pb concentration
(0.85±0.03) was maximum whereas Cd (0.16±0.03) found lowest in Qarshi. Versatile
contains maximum Pb (1.34±0.02) while lowest Cd (0.12±0.02). In Al-hayat Cu
concentration (1.23±0.03) was maximum while Pb (0.11±0.03) was lowest. Young’s
honey contains maximum Ni (2.41±0.01) while lowest Mercury (0.16±0.03). Ni
(1.25±0.02) was found maximum and Mn (0.14±0.03) lowest in Pak-salman, whereas
in Langnese Hg concentration (0.71±0.03) found maximum while Cd (0.13±0.02) was
lowest. The contamination level of aflatoxins (B1, B2, G1 and G2) was also evaluated
in both types of honey. Minimum level of aflatoxins were detected in branded and
unbranded honey sample are B1and B2 such as (2.14, 1.25) and maximum
concentration are (2.33, 2.15) respectively.
It is concluded that the unbranded honey had more contamination of antibiotic
residues as compared with branded and natural honey. All the branded and unbranded
and natural honey samples evaluated showed antioxidant activity. Natural honey
samples presented better activity as compared to branded and unbranded samples.
Thus specifically honey could be used as alternative natural antioxidant in different
formulations for food and pharmaceutical industries.
It is evident from this study that, processing of honey may effects the phenolic acid
contents of honey. Honey has effective inhibitory affects and has antimicrobial
activities. Thus may be utilized in many food as well as netraceutical products for
human consumption.
54
The unbranded honey samples are also good source of nutrients and valuable
phytochemicals as compared to branded samples. Due to lack of information available
on chemical composition and phytochemicals in these honeys and their role in diet,
the assessment was carried out on the basis of nutritional quality. So these available
honeys can be utilized in various food products as well as in herbal formulations
It is further evident from the study that beneficial carbohydrates contents were
found in all natural and farm honey’s samples. The direct heating much increases the
H.M.F concentration in honey samples as compared to the oven. So it necessary for
beekeepers to use the electric oven in the processing of honey and avoid from direct
heating of honey. So, it is concluded that contaminants are less as compare to the
reported values so mostly the honey produces in Khyber Pakhtunkhwa are good for
use and export can be enhanced.
GENERAL INTRODUCTION
Honey is a sweet and viscous fluid produced by honeybees from nectar of
flower’s [1]. It is a mixture of sugar and other compounds. Honey is mainly fructose
(about 38.5%) and glucose (about 31.0%), making it similar to the synthetically
produced inverted sugar syrup which is approximately 48% fructose, 47%
glucose, and 5% sucrose [2-3], remaining carbohydrates include maltose, sucrose,
and other complex carbohydrates [4]. Apart from this, many polyphenols such as
pinobanksin, quercetin, chrysin, caffeic acid, calangin, apigenin, acacetin,inocembrin
and kaempferol have been reported as pharmacological agents of honey and used for
the treatment of cancer [5-6]. Nutritive sweeteners are not only the main source of
55
honey but also have some contents of vitamins and minerals [7]. The production of
specific honey is dependent on the availability of pollens to honeybees [8].
Honey is used as food in cooking, baking, as a spread on breads, and as an
addition to various beverages such as tea and as a sweetener in some commercial
beverages. It can be used as instant energizer as it contains sugar which is quickly
absorbed by our digestive system and converted into energy [9]. Honey is also used
for different medicinal purposes since ancient times. It exhibits an inhibitory effect on
yeast, fungi, leishmania, mucocutaneous injuries such as genital lesions, superficial
skin burns, post operation wounds, gastrointestinal, cardiovascular, inflammatory and
neoplastic states [10].
The antioxidant (scavenging) activity of honey is due to the presence of
various compounds such as phenolic compounds, vitamins, amino acids, flavonoids,
carotenoid’s, enzymes peroxides, catalase and glucose oxidase [11]. Free radicals lead
to oxidative damage in many molecules, such as lipids, proteins and nucleic acids
[12]. The phenolic compounds significantly contribute to the health of human by
blocking the production of free radicals in the body. These compounds are mainly
available in waxes, pollens and propolis [13]. Honey shows antiulcer, immune-
stimulant, antifungal, antimicrobial, anti-inflammatory and regenerative activity on
human due to phenolic compounds [14]. During emotional, intellectual and physical
stress, honey shows antidepressant activity [15].
Beside the usefulness of honey, there are certain harms like heavy metals and
pesticides contaminants exist in honey making it infected [16]. Bees transport these
contaminants to beehives from plants, soil and water [17]. There are different diseases
caused by bacteria i.e. European Foulbrood and American Foulbrood which infect the
honey bees [18]. For curing these diseases, the bee keepers mostly use different
56
antibiotics such as ampiciline,oxytetracycline, streptomycine, nitrofurone,
sulfonamides, and cholorophenicoles whichcause the presence of residues and active
metabolite of drugs in honey [19]. In some European countries, use of antibiotics is
illegal for beekeeping [20]. Antibiotic residues have toxic acute and chronic effects on
human health, and reduce the efficacy and quality of honey [21].
The present study is designed to investigate the antibiotic residues and their
metabolites in honey of Khyber Pakhtunkhwa Pakistan. The quality parameters,
nutritive as well as pharmacological aspects, like phytochemical composition,
antioxidant, antimicrobial activities and contaminants are to be analyzed.
57
CHAPTER - 1
1. INTRODUCTION
1.1. Honey
Honey is a sweet and viscous fluid produced by honeybee’s from nectar of
flowers [1]. The Codex Alimentarius commission defines honey as “The natural sweet
substance produce by honeybee’s from the nectar of flower or from secretions coming as of
living organisms’ feeding on plants, that bee’s gather, transform and combine with specific
ingredients, store and leave to ripen in the combs of the hive [2]. Honey was also defined as
a pure natural product which does not include any other substances, like water or
sweeteners. This definition has been widely accepted by the food regulation of most
countries, including Pakistan (Figure 1.1, a).
Figure 1.1, a: Honey
www.dreamstime.com
58
1.2. Production and Consumption of Honey
The annual worldwide production of honey is about 1.4 million tons estimated.
Honey is mostly produced in Asia, accounting for about 40% of the global production. China
is the chief producer of honey, producing approximately 0.3 million tons honey annually
(Figure 1.2, a) [3].
Figure 1.2, a: Honey production by country [4].
59
Figure 1.2,b: World per year honey consumption
Developing countries consumed generally higher amount of honey such as Egypt, Brazil,
China, India and Argentina, is estimated to be (0.1 – 0.2) kg per capita. However, the per
capita honey consumption is resolute the cultural influence. It is not following the richness
of the countries (Figure 1.2, b).
In the European Union, Greece is the chief honey consumers with 1.8 kg /capita, UK
is the lowest consumers 0.4 kg/capita, while the intermediate range consumed in Germany
with (1.5 kg), Hungary, France, Spain and Italy are (0.6 - 0.9 kg) [3]. In India, 40% of the total
organized sector honey producers. Every year production of honey is about 65000 tons.
Annually export of honey is about 25000 tons, which is more than forty two countries,
together with European Union, United State and the Middle East [5].
In Pakistan beekeeping is a beneficial business. More than 7000 beekeepers are now
raring exotic species; modern bee hives like (Apis mellifera). Pakistan produces 7500 metric
tons honey annually. About 300,000 colonies of honey bee’s are present in Pakistan.
Favorable climate condition and bee flora provide tremendous opportunity for the
development of beekeeping. More than 1,000,000 colonies of honeybee’s flora are present
60
in all provinces, including northern areas, federally administrated tribal areas (FATA) and
Kashmir [6].
1.3. History of Honey
In Georgia, the Archaeologist have found honey remain on the interior surface of
clay vessels unearthed an prehistoric tomb, dating back to some 4700 to 5500 years ago [7].
The greater honey guide bird, guides humans to wild bee hives and this performance may
have evolved with early hominids [8]. In ancient Georgia honey was packed for peoples
journeys into the afterlife and more than one type too along for the trip were linden
meadow- flower verity and berry [9]. In historic Middle Eastern and Egyptian peoples used
honey for dead embalming. Honey was used to sweeten cakes, biscuits and many other
dishes in ancient Egypt [10]. The remedial and religious use of honey in ancient India is
recognized in both the Ayurveda and the Vedas texts, which were both, packed together at
least 4000 years ago [11].
In Islamic medical system honey is considered as a healthy drink. Honey has also
been cited in the Holy book of Muslim (Quran) (Section 16 Verse 68-69) indicated the
medicinal properties many centuries ago. The holy Quran vividly illustrated the prospective
beneficial value of honey; And the Lord idea the bee to make its unit in hills, on trees, and in
(men’s) habitations; then to eat of all the produces (of the earth) and find with skill the
spacious paths of its Lord: there issues from within their bodies a drink of varying colors,
wherein medicinal for men: verily in this is a sign for those who gave thought;” furthermore,
the Muslim prophet Mohammad (SA) advised the use of honey for the cure of diarrhea [12].
Almost 1000 years ago, the great Iranian physician and scientist ‘Avicenna’ had suggested
honey is one of the finest remedies in the cure of tuberculosis [13].
1.4. Physical Properties of Honey
61
Freshly extracted honey is a sticky and glutinous liquid. Its viscosity depends on
water contents, its composition and particularly verities of substances. Honey has a variety
of important qualities in count of composition, flavor and taste. The foaming characteristic
of honey usually depends on its viscosity. Another property of honey is its hygroscopicity
which described the ability of honey and embrace moisture from environment. Surface
tension of honey change due to its origin and colloidal substances [14]. Honey color varies
from colorless, clear to black, dark amber. The different coloration of honey is mainly all
shades of amber and yellow. Color change with storage condition, age and botanical origin,
but clarity or transparency depends on the quantity of suspended particles such as pollens.
Less frequent honey color are bright yellow (sun flower), radish (chest nut), greenish (honey
dew) and grayish (eucalyptus), due to crystallization honey turns lighter in color because the
glucose are white crystal [15]. Honey crystallization consequences from the formation of
monohydrate glucose crystals which differ in dimension, quality, numbers and shape with
the storage condition and composition of honey. The higher glucose content and the lower
water in honey, faster the crystallization [16].
The composition of honey varies from crop to crop and season to season. From
various locations the same was correct for the same type of honey. As formerly known, light
honey is lower nitrogen and ash contents then darker honey. Average regions of the United
States showed results that eastern and southern honey was darker than usual while with
northern central and intermountain honeys were lighter in color. The intermountain honey
showed high granulation affinity while South Atlantic State honey has least tendency to
granulate. The average of moisture were high in north central honey [17]. The constituents
of honey are mostly expressed in percent. Natural comb moisture of honey is that which
remnants after the nectar refining. The moisture quantity is an important function factor of
refining, together with weather conditions and original moisture of the nectar. One
characteristic of honey is moisture that influences its granulation property and quality. After
62
extraction of honey, depending on storage conditions, moisture content may be changed.
The honey buyers and beekeepers know that the moisture content of honey varies
extensively and it may range between 16 to 25 percent [18].
Honey can be characterized according to its geographical origin. Regional variation
of honey were reported by many scientist in the physiochemical property of honey samples,
such as the enzymes activity, ash content, electrical conductivity, pH and Hydroxy Methyl
Furfural (HMF) [19]. The colors variations of honey are exclusively due to the plant source,
heat also change the color of honey by darkening action. Color of honey varies a continuous
range from pale yellow through amber to a darker red to black [20].
The major portion of honey consist of sugar a very concentrated solution of several
sugar result in the physical property of honey like high viscosity, high density, affinity to
absorb air moisture and protection from some types of spoilage. Honey have very high
granulation tendency. Due to this special character honey is different from other
sweeteners [21, 22].
1.5. Chemical Composition of Honey
Natural honey contain about two hundred substances, which consist of not only
highly concentrated solution of sugars, but also the complex mixture other substances like
saccharides, amino acids, peptides, proteins, enzymes, polyphenols, organic acids, vitamins,
carotenoid and minerals (Figure 1.3, a) [23]. Sugars are the chief constituents of honey,
containing about (95%) of its dry weight [24]. Honey mostly contain fructose 38.5% and
glucose 31%, making it similar to the synthetically produced inverted sugar syrup which
is about 48% fructose, 47% glucose and 5% sucrose [25]. It is a mixture of carbohydrates,
such as fructose 25 to 45%, glucose 25 to 37 %, maltose 2 to 12% and sucrose 0.5 to 3 %.
Honey also contains water content 15 to 18% and some trace amount of other sugars
63
depending on floral source. Some range of nutritiously essential elements and is high-
viscous liquid [26]. Honey contains roughly 0.5% proteins whilesome honeys can be over
1000 µg/g [27]. Almost all of physiologically essential amino acids are present in honey [28].
The primary amino acid is proline, contributing 50-85% of the total amino acids [29].
Figure1.3, a: Chemical composition of honey
The presence of enzymes in honey is a unique characteristic due to which it is
different from all other sweetening agents. These enzymes originate from the yeasts, pollen,
bee nectar and micro-organisms present in honey. Enzymes are complex protein materials
that under mild conditions bring about chemical changes. Some of the most significant
honey enzymes are catalase, diastase, phosphatase, glucose oxidase and invertase. Enzymes
Carbohydrates
(Fructose and sucrose)
Plant hormones
(Abscisic acid and
phaseic acid)
Organic acids
(Malic acid and
fumaric acid)
Minerals
Potassium,
calcium,
magnesium and
sodium
Vitamins
(Riboflavin and
thiamine)
Processed
derived
compounds
(Quebecol)
Polyphenols
(Lignin’s)
Amino acids
(Threonine, arginine and
proline)
Composition of Honey
64
in honey can be degraded by heating process [30]. The main sources of honey are not only
nutritive sweeteners but also have some contents of minerals [31].
Investigations have shown that a wide range of trace elements are present in honey,
including (Al, Ba, Bi, Co, Cr, Mo, Ni, Pb, Sn, Ti), as well as minerals (Ca, Cu, Fe, K, Na, Mg, Mn,
Zn) [32]. Among them, the main mineral element is potassium,while copper found as lowest
[33]. Honey contains vitamins such ascorbic acids vitamin C, thiamin (B1), riboflavin (B2) and
pyridoxine have also been reported, is very less amount in honey [34].
Honey contains various polyphenols such as acacetin, apigenin, caffeic acid, crysin,
inocembrin, quercetin, kaempferol and pinobanksin as pharmacological agents and used for
the curing of cancer [35]. The production of specific honey is dependent on the accessibility
of pollens to honeybee’s [36].
1.6. Uses of Honey
Honey is used as food in backing, cooking, spread on breads and addition to various
beverages such as tea and a sweetener in some commercial beverages. It can be used as
instantaneous energizer because it contains sugar which is readily absorbed by our digestive
system and changed into energy [37]. Honey used for different medicinal purposes science
ancient times. It shows an inhibitory effect on leishmania, fungi, yeast and mucocutaneous
injuries such as post operation wound, genital lesions, cardiovascular, inflammatory,
superficial skin burn, gastrointestinal and neoplastic states [38].
Honey has been used to treat wounds for thousands of years. It was displaced from
use after the arrival of antibiotics. Now the antibiotic era is coming to an end and honey is
being rediscovered. On other hand the use of honey without awareness of ancient
perception and without using the right honey may consider it fable as they may not get good
65
result. The used of honey appropriately will get good result which seems to be miraculous.
Honey is better and effective for healing like modern pharmaceutical products [39].
To achieve these many beneficial effects it is necessary to keep honey in contact
with wound bed. Secondary dressing can be used on non-exudative wound. But when there
is exudates, honey impregnated absorb dressing are needed. The frequent changes of these
are important when there are numerous exudates flushing the honey out of the dressing.
Honey impregnated alginate fiber dressing, which convert to a soft gel are bitter but have
limited exudates absorbing capability (Figure 1.4, a).
66
Figure 1.4,a: Traditional and modern uses of honey [40].
A new form of gelled honey dressing like a hydrocolloid has a very large capacity for
absorbing exudates keeping the honey in contact with the wound bed [41, 42]. Honey was
also used in diabetes associated with considerably lower glycemic index as compare to
sucrose or glucose in normal diabetes. In earlier observation it was found that honey
motivate insulin secretion, raise hemoglobin concentration, decrease blood glucose level
Uses of Honey
Traditional
Modern
Muslims: Used honey
for the treatment of
diarrhea and
tuberculosis
Indians: Used hone for
Irritating cough, teeth
and gum protection, skin
disorder, wounds, burns,
cardiac pain, lungs,
anemia, palpitations and
eye sight.
Egyptian: Used honey
for embalming the dead
Treatment of urinary tract
infections, bacteriostatic,
bactericidal, radical surgery for
carcinoma of the breast and
varicose veins, gastritis
gastrointestinal tract infections,
gastriculceration, healing of
peptic disease
Antifungal activity, inhabit toxin
production, Inhibit rubellavirus
activity, ophthalmological
conditions like blepharitis,
keratitis, conjunctivitis, corneal
injuries, chemical and thermal
burns to eyes
Diabetic use: stimulates insulin
secretion, decrease blood
glucose levels, elevates
hemoglobin concentration and
improves lipid, anti-
inflammatory,as antioxidants:
controlled free radicals, its
anticarcinogenic,cardiovascular,
asthmatic heart diseases, chest
pain, fatigue, high nutritional
energy, respiratory ailments,
measles,period pains, postnatal
disorders, male impotence and
protect DNA damage.
Greece: For baldness,
contraception, wound
healing, laxative action,
cough, sore throat, eye
diseases, topical
antisepsis prevention
and treatment of scars
67
and improve lipid profile [43]. Humans use honey from ancient time is about some 8000
years ago as depicted by Stone Age paintings [44]. In ancient Greeks, Chinese, Romans and
Egyptian employed honey for gut diseases and wounds. The ancient Egyptians used honey as
a contemporary ointment and for dead embalming [45]. In Greece, the honey beverage
containing grape juice is used for nervous disorders, pain, thirst, acute fevers, sore throat,
cough, topical antisepsis, eye disease, treatment and prevention of scars [46].
In Indian system honey is Ayurveda. Meaning ‘knowledge of life’ honey is used in the
treatment of irritating cough, Cardiac pain, palpitation, burns, wounds, anemia and all
imbalances of the lungs [47]. Natural honey can play vital role in the treatment of vertigo,
fatigue and chest pain. It’s also useful in tooth extraction pain and infection. In central
Burkina Faso, it is also used for treatment of measles, postnatal disorders, respiratory
ailments due to its anti-inflammatory, antibacterial and anti leishmania effects [48]. Honey
showed positive effect on blood level of minerals, enzymes, hematological indices and
endocrine system. During primary and secondary immune response, it exited the antibody
production against thymus-dependent and thymus-independent antigens [49].
1.7. Classification of Honey
Honey is classified by its floral source, processing and packaging used. Honey is also
graded on its optical density and color by United State Development and Agriculture
Standard, graded on a scale called Pfund scale, which range from 0 for “water white” honey
to more than 114 for “dark amber” honey [50].
68
Figure 1.5, a: Classification of honey
Generally, honey is classified by the floral source of the nectar from which it was made
(Figure 1.5, a). Honey can be from specific type of flower nectar or can be blend after
collection. The pollens in honey is definite to floral source and therefore of origin. The
melisso palynological and rheological properties of honey can be used to identify the major
plant nectar source used in its production [51]. Most commercially available honey is
blended, meaning it is mixtures of two are more honeys differing in color, floral source,
geographical origin, flavor and density [52].
Monofloral honey make mostly from the nectar of one variety of flower. Various
monofloral honeys have a typical color and flavor because of differences between their main
nectar sources. Typical examples of North American monofloral honey are tupelo, orange
blossom, clover, sage, blueberry, buckwheat, tupelo, sourwood and fireweed [53]. Polyfloral
honey also known as ‘wildflower honey’ derivative from several varieties of flowers nectar.
The flavor may change from year to year and smell and the taste can be more and less
excessive, depending on which blooming is prevalent (Figure 1.5, b) [54].
69
Figure 1.5,b: Classification of honey on the basis of flowers sources and processing
As an alternative of taking nectar, bees can take honeydew, the sweet secretions of
aphids or other plant sap-sucking insects. Honeydew honey is very dark brown in color, with
a rich delicate scent of fig jam and is not sweet as nectar honey [55]. North California and
Germany black forest is well known source of honeydew honey as well United States,
Bulgaria and Tara mountains in Serbia. In Greece, pine honeydew honey constitutes 60 to
65% of the annual honey production [56].
70
1.7.1. Classification Based on Processing
Normally, honey is bottled in its familiar liquid form. But it is sold in other forms and
can be passed to a variety of processing methods. Granulated or candied crystallized honey
is honey in which a few of the glucose content suddenly crystallized from solution as the
monohydrate. Honey that has crystallized or commercially purchased crystallized can be
return to a liquid state by warming [57]. Pasteurized honey is honey that has been heated in
a pasteurization process which required temperature 72°C or higher. Pasteurization
destroyed yeast cell. It also liquefies any micro crystals in the honey, which delays the onset
of visible crystallization. Heat affects the level of Hydroxy Methyl Furfural (HMF), taste,
fragrance and manifestation (darken the natural honey color) (Figure 1.5, c) [58].
O
O
OH
Figure 1.5, c: Hydroxy Methyl Furfural formation
71
Honey Processing Plant
Details of honey processing plant
A: Homogenizer F: Falling film evaporator
H: Elictric heaters S: Filters
PC: Processing coil P1: Pump for raw honey
P2: Pump for processed honey P3: Vacuum pump
P4: Hot water circulating pump V2: Hot water generator
V1: Primary heating vessel jacketed with fins V4: Condensate receiver
V3: Vapor separatorV5: Settling tank C2: Honey cooler
C1: Vapor condenser
S1: Trough screen filter = 60-304 mesh
S2: Online screen filter = 100-304 mesh
Note: Yellow line indicate flow of honey
Blue line indicate vacuum and green line
indicate flow of water
72
S3: Online primary filter (polypropylene bag 10 micron)
S4: Online secondary filter (resin bonded cartridge 5 micron)
Figure 1.5,d: Honey processing plant [59].
Figure 1.5,e: Processing of honey extraction [60].
Raw honey are those which exist in the beehives or as obtained by extraction,
straining or settling, without adding heat (although some honey that has been
‘”cleanly processed” is often labeled as raw honey). Raw honey contains small
particles of wax and some pollen (Figure 1.5, d; 1.5, e). Some allergy suffers try
using raw local honey to build up a tolerance to the pollens in the air. However, hay
Pre-
Heatin
g
Micro
Filter
Processing
Tank
Feeding
Tank
Honey
Pump
Condensate Moisture
Reduction
Hot Water
Generator
Vacuum Pump Condensate
Collection Cooling
Bottling Settling Tank
Centrifugal
Pump
Honey processing steps
1. Liquefaction
2. Pre-Heating and Straining
3. Microfiltration
4. Inactivation of Yeast Cells (processing)
5. Vacuum Evaporation
6. Cooling
7. Bottling
Liqueficat
ion
Step 1 Step 2 Step 3 Step 4
Step 6
Step 5
Step 7
73
fever is normally caused by pollen in the air, which is mostly from weeds, grass and
trees, rather than flowers [61].
Figure 1.5f: Tools of processing and extraction of honey [62].
Filter honey is honey of any type that has been filtered to the extent that all or most
of the fine particles, air bubbles, pollen grain, or other materials normally found in
suspension, have been removed (Figure 1.5, f). The process usually heats honey 66°C to 77
°C to more easily pass through the filter. Filtered honey is extremely clear and will not
crystallize rapidly, making it preferred by the supermarket trade [63]. Strained honey has
been passed through a mesh martial to remove particulates material (pieces of propolis wax
and other defects) without removing pollens, enzymes or minerals [64].
Creamed honey, it’s also called spun honey, whipped honey, honey fondant, candied
honey, churned honey, and in the (UK) set honey has been processed to controlled
crystallization. Creamed honey contains a large number of small crystals, which prevent the
formation of larger crystals that can occur in unprocessed honey. The processing also
produces a honey with a smooth, spreadable uniformity [65]. Chunk honey is packed in wide
mouth container consisting of one or more pieces of comb honey immersed in extracted
liquid honey [66].
74
Ultrasonicated honey has been processed by ultra-sonication, a non-thermal alternative
process for honey. When honey is exposed to ultra sonication, most of the yeast cell is
destroyed. Those cells that survive sanction generally loss their ability to grow, which reduce
the rate of honey fermentation considerably. Ultra sonication also remove existing crystal
and inhabits further crystallization in honey ultrasonically aided liquefaction can work at
significantly lower temperature of about (35°C) and can reduce liquefaction time to less than
30 seconds [67]. Dried honey has the moisture extracted from liquid honey to create
completely solid, non-sticky granules. This process may or may not include the use of
anticaking and drying agents. Dried honey is used in baked goods [68].
Comb honey is still in the honeybees wax comb. It traditionally is collected by using
standard wooden frames in honey supers. The frames are collected and the comb is cut out
in chunks before packaging. As in alternative to this labor-intensive method, plastic rings or
cartages can be used that do not required manual cutting of the comb, and speed packaging.
Comb honey harvested in the conventional manner is also referred to as “cut- comb honey”
[69]. Honey decoctions are made from honey or byproduct of honey which have been
dissolved in water, then reduced usually by mean of boiling. Other ingredients may then add
(like abbamele has added citrus). The resulting product may be similar to molasses [70].
1.8. Species of Honey Bee’s
Stingless bee’s, belong to the family Apidae, are a large group of bee’s about
500 species, sometimes called meliponines, comparing the tribe meliponines [71].
They are closely related to common honeybee’s, orchid bee’s, bumblebee’s and
carpenter bee’s. The common name is slightly misleading, as bee’s of other species
and male bee’s such as in the family Andrenidae cannot sting. Meliponines have
stinger but they are highly reduced and cannot be used for defense [72].
75
Figure 1.6,a: Stingless bees
Stingless bee’s can be found in most tropical and subtropical region of the world such
as Africa, South Asia, Australia, tropical central and South America, including
Madagascar [73]. All year round the stingless bee’s are active but some species are
less active in cooler weather [74]. Stingles bee’s frequently form nest in hollow trunk,
underground cavities and tree branches (Figure 1.6, a). Mostly the beekeepers keeps
the bees in their original log hives or transfer them to a wooden box or put them in
bamboos, flowerpots, it easier to control the hive [75, 76]. Bumble bee, belong to the
family Apidae, is a member of the bee genus Bombus. They are more than 250 species
76
found primarily in higher altitude or latitude in the northern hemisphere, although
they also arise in South America, New Zealand and Tasmania. Bumblebee’s have
round bodies enclosed in soft hair long branched setae, called pile, making them
appear and feel nebulous. They have warning coloration, regularly in bands, in
combination of black, red, white, orange and yellow [77].
Figure 1.6,b: Bumble bees
Bumblebee’s are personalized to form a pollen basket, an uncovered shiny concave
surface, surrounded by a fringe of hair used to transport pollens (Figure 1.6, b). They
form colonies with a single queen; colonies are smaller than those of honey bee’s
consisting often of fewer than 50 individuals in the nest. Female bumblebee’s can
frequently sting but usually ignore humans and other animals. Bumblebee’s can
regulate their body temperature called heterothermy [78]. Due to which few species
(Bombus alpines and Bombus Polaris) range into very cold climate. Bumblebee’s
77
generally visit flowers a habit known as pollinator. While foraging, bumble bee’s can
reach ground speed up to 15 meters per second or 54 kilometer per hour [79].
Four species of honey bees are found in Pakistan. Three species are native and
one is imported and established in Pakistan. The indigenous species are Apis cerana,
Apis florea and Apis dorsata (Figure 1.6, d; 1.6, e and 1.6, f). The occidental species
is Apis mellifera (Figure 1.6, c). In different ecological areas of countries these
species are present [80]. In Pakistan, Apis mellifera was introduced in 1977. The Apis
florae commonly known as “choti maki” and Apis dorsata common name is
“doomna” are wild in nature and makes hives in open palces. Both have about 12,000
to15, 000 colonies, respectively [81].
Figure 1.6,c: Apis mellifera bee’s
78
Figure 1.6,d: Apis florae bee’s
Figure 1.6,e: Apis dorsata bee’s
79
Figure 1.6,f: Apis cerana bee’s
1.9. Diseases of Honey Bee’s
1.9.1. American Foul Brood Diseases
American foulbrood is caused by a spore-forming bacterium Paenibacillus larva.
Young honey beelarvae become infected when they consume P.larvae spores in their food.
The spores germinatein the gut; bacteria then move into the guttissues, where they multiply
enormously innumber [82]. Infected larvae normally die after theircells are sealed. Millions
of infective spores areformed in their remains, which dry to form‘scales’ that adhere closely
to the cell wall and can not easily be removed by bee’s [83]. Consequently brood combs
from infected colonies are inevitably severely contaminated with bacteria spores (Figure 1.7,
a). If the scales go unnoticed and infected combs are subsequently used or moved from
colony to colony during routine beekeeping management, then infection has the potential to
spread quickly [84].
80
Figure 1.7,a: American foulbrood diseases [85].
1.9.2. European Foul Brood Diseases
European foulbrood is caused by the bacterium Melissococcus plutonius. The
bacteria multiply in the mid-gut of an infected larva, competing with the larva for its food
[86]. They remain in the gut and do not invade the larval tissue; larvae that die from the
disease do so because they have been starved of food. This normally occurs shortly before
their cells are due to be sealed [87]. Subsequently other species of bacteria may multiply in
the remains of dead larvae (Figure 1.7, b). Such ‘secondary invaders’ include Paenibacillus
alvei, Enterococcus faecalis, Brevibacillus laterosporus and Lactobacillus Eurydice [88].
Dead pupae
Irregular and sunken brood
Americans Foul Brood (AFB) Disease
Cause: Paenibacillus larva.
Most widespread and destructive disease
It affects honey bees drones, queens and
workers
Infection: Gut
Symptoms: turn dark brown and later
changes to sticky mass and produce foul like
smell, infected larvae darken and die
Infected stage: larvae
Management use: Oxytetracycline and tylosin
81
Figure 1.7,b: European foulbrood disease [89, 90].
1.10. Antibiotics
Antibiotic is an agent that ‘either kills or inhabits the growth and development
of microorganism. Antibiotics are medicines used to protect the health and benefit of
humans and animals. It abolishes or inhabits the growth of microorganisms such as
fungi, protozoa or bacteria. The word antibiotic formerly used to every agent with
biological activity against living organisms; however ‘’antibiotic” now refers to
substances with antifungal, antibacterial or anti- parasitical activity. There are
presently about 250 different chemical units registered for use in medicine and
veterinary medicine [91].
European Foul Brood (EFB) Disease
Cause: Melissococcus plutonius, bacillus pluton
(bacterium)
Infection: Mid gut
Symptoms: The infected larvae turn yellow and
then brown, the tracheal system becomes visible.
Larvae die in coiled stage
Causing fuel smell, cells are poorly capped and
mixed with normal cells
Infected stage: Larvae
Management: Use oxytetracycline hydrochloride
Infected larvae yellow brown
Larvae coiled stage
82
1.10.1. Brief History of Antibiotics
In 1942, Selman Waksman and its collaborators first time used the term
antibiotic in journal articles to explain any substance produce by microorganism that
is aggressive to the growth of other organism in high dilution. Before the early 20th
century, treatments for infection were based mainly on medicinal legends. It was
described about 2000 years ago that the mixture with antimicrobial properties were
used in treatments of infections (Figure 1.8, a) [92].
The French bacteriologist Jean Paul Vuillemin introduces the word
“antibiosis” meaning “against life” as an expressive name of the phenomenon showed
by these early antibacterial drugs. Antibiotic was first described in 1877, bacteria
when Robert Koch and Louis Pasteur observed that an airborne bacillus could inhabit
the growth of Bacillus anthraces. Synthetic antibioticchemotherapy as a science and
development of antibacterial begins in Germany with Paul Ehrlich in the late 1880s
(Figure 1.8, b) [93].
Figure 1.8,a: Discovery of different antibiotics from 1940 to 2000 [94].
83
Figure 1.8,b: Brief history of antibiotics [95].
In 1928, Alexander Flaming discovered antibacterial compound, named penicillin
(Figure 1.8, c). In 1932, Gerhard Domagk and research team developed the first
commercially available antibacterial drug sulfonamide at the Bayer Laboratories of
the IG Farben conglomerate in Germany [96].
Figure 1.8,c: Penicillin structure [15].
In 1939, with the beginning of World War II, René Dubos reported the
discovery of the first naturally derived antibiotic, tyrothricin a compound of 80%
tyrocidine and 20% gramicidin from B.brevis. However due to toxicity gramicidin
Brief History of Antibiotics
1928- Penicillin discovered by Fleming
1932- Sulfonamide antimicrobial activity discovered by Erlich
1943- Drugs companies begin mass production of penicillin
1948- Cephalosporin’s precursor send to oxford for synthesis
1952- Erythromycin derived from Streptomyces erythreus
1956- Vancomycin introduced for penicillin resistant streptococcus
1962- Quinolone antibiotics first discovered
1970s- Linezolid discovered but not pursued
1980s- Fluorinated quinolones introduced, making clinically useful
2000s-Linezolid introduced into clinical practice
84
and tyrocidine could not use systematically. In 1942, Chain and Florey succeeded in
purifying the first penicillin, but it did not widely available outside the military before
1945. Dorothy Crowfoot Hodgkin determined the chemical structure of penicillin in
1945. Until 1940s, despites this discovery, penicillin was not made available as the
first true antibiotic [97].
Figure 1.8,d: Streptomycin structure [16].
Albert Schatz reported the isolation of the first amino glycoside antibiotics
streptomycin from (Streptomyces griseus) in 1943. Streptomycin was the first
antibiotic to be effective against tuberculosis (TB) (Figure 1.8, d) [98].
Figure 1.8,e: Chloramphenicol structure [99].
85
Chloramphenicol was subsequently used to treat typhus and subsequently typhoid
fever but its use started to decline in 1960 due to its ability to induce aplastic anemia,
bone marrow suppression and the gray syndrome (Figure 1.8, e) [100]. Antibacterial
substances isolated from a strain of Cephalosporium acremonium by Giuseppe Brotzu
in the mid-1940s, Florey and his co-workers in Oxford discovered the cephalosporin
family of β-lactams which were very active against a wide spectrum of bacterial
infections with a very low toxicity [98].
The isolation of (6-APA) paved the way for a large-scale production and
marketing of semi-synthetic penicillin during the 1960s, including ampicillin,
methicillin, flucloxacillin, amoxicillin, ticarcillin and carbenicillin, followed later by
mezlocillin, azlocillin, piperacillin and mecillinam [101]. Chlortetracycline was the
first member of the tetracycline group to be isolated by Benjamin M. Duggar in 1947;
oxytetracycline then soon followed [102].
Figure 1.8,f: Oxytetracycline structure [103].
Tetracycline are the second most commonly used antibiotics after the
penicillin due their activity against a long list of infections and because they are
relatively cheap to produce (Figure 1.8, f) [104]. The 1970s, which witnessed an
increase in the resistance among Gram-negative bacilli, saw the introduction of
86
amikacin, a semi synthetic derivative of kanamycin [105]. In 1962 Lesher and his co-
workers recognized nalidixic acid, a by-product of chloroquine production [106],
which became the first quinolones antibiotic to be developed. The first-generation
quinolones were active against aerobic Gram-negative bacillary infections, especially
those found in the human urinary tract. This limited activity against aerobic Gram-
negative bacteria was enhanced (1000-fold) in 1980s when the second-generation
fluoroquinolones were introduced [107].
1.10.2. Classification of Antibiotics
Antibiotic can be grouped by both their mechanisms of action or by chemical
structure. They are frequently complex molecules which may possess different
functionalities within the same molecule. Therefore, under different pH conditions antibiotic
can be cationic, anionic neutral or zwitterionic. They are divided into different sub-groups
such as amphinicoles, amino glycosides, β-lactams, tetracycline’s, aminofluoroquinolones
and macrolides (Figure 1.8, g) [108].
87
88
Figure 1.8,g: Classification of antibiotics their uses and side effects on humans
β-lactams antibiotics have a β-lactams ring nucleus with a hetero atomic ring
structure, containing of three carbons atom and one nitrogen atom, used to cure bacterial
infections by attacking the bacterial cell wall i.e. Pencillines, amoxicillin and ampicillin [109].
Amphinicoles are antibiotics group with a phenyl propanoid structure. Theirfunction
by blocking the enzyme peptidyl transferase on the bacterial ribosome subunit (50S) i.e.
Azidamphenicol, chloramphenicol, florfenicol and thiamphenicol [110].
Tetracycline’s antibiotic with four (“-tetra-“) hydrocarbons ring (“-cyclic-“) origin
(ine) define as “a substance having octahydrotetracene-2-carboxamide skeleton of poly
ketoses ” used for the treatment of bacterial brood disease i.e. tetracycline, chloro
tetracycline and oxytetracycline [111].
89
Macrolides are lipophilic and basic antibiotics among 14 member macro cyclic lactones ring
connected by glycosidic linkages and are effective against wide variety of Gram negative and
positive bacteria used for the treatment of infectious diseases in cattle, swine, poultry and
sheep i.e. erythromycin, lyncomycin and tylosin [112].
NH2
OO
OH
NH
CH3
CH3
OH
O
OH
NH2
CH3
NH2
CH3
Figure 1.8,h: Gentamycin structure [113].
Amino glycosides composed of an amino cyclitol ring attached to two are more
amino sugar connected by a glycoside link used for the bacterial brood disease treatment i.e.
neomycin, gentamycin and streptomycin (Figure 1.8, h) [114]. Fluoroquinolones contain a
fluorine atom linked usually to the 6-position of central ring system, and used as growth
promoters up to date and reliable data on antibiotic utilization for humans and animals is
not extensively obtainable e.g. enrofloxacin, norfloxacin and ciprofloxacin [115].
1.10.3. Antibiotics Allowed in Beekeeping
Beekeepers use comparatively high doses antibiotics, as useful agent to treat clinical
infections bacterial brood diseases or they may be administered at low, sub curative doses
as ‘growth promoters’. The antibiotics uses is less labor intensive and more advantageous in
beekeeping [116].
90
A list of product permitted for use worldwide for combating bee diseases (Table 1) suitable
every day intake [117], recognized also by the joint WHO/FAO used against mites and
antibacterial substances such as tetracycline, sulfonamide, erythromycin, streptomycin and
tylosin used in the healing of bacterial brood diseases. (MRLs) have been recognized for all
food producing species for tetracycline’s and sulfonamides but there is no (MRLs) for honey
[118].
Table 1: List of approved products in apiculture
91
SUBSTANCES MAJOR
APPLICATION
PROPRIETA
RY
PRODUCT
ADI (MG/KG
BETWEEN PER DAY)
JECFA JMPR
ACRINATHRINE PESTICIDES/ACARI
CIDE
YES
AMITRAZ PESTICIDES/ACARI
CIDE
YES 0-0.01
BROMOPROPYLATE PESTICIDES/ACARI
CIDE
YES 0-0.03
CHLOROBENZILATE PESTICIDES/ACARI
CIDE
NO 0-0.02
COUMAPHOS
(PERIZIN)A
PESTICIDE YES
CYMIAZOLE
HYDROCHLORIDE
(APITOLE)
PESTICIDES/ACARI
CIDE
YES 0-0.03
FENPROXIMATE PESTICIDE YES
FIPRONIL PESTICIDE NO 0-0.0002
FLUMETHRIN(BAYVARO
L)
PESTICIDE YES 0-0.004
CHLORTETRACYCLINE VETERINARY DRUG NO 0-0.003
FUMAGILLIN PESTICIDE YAS
LACTIC ACIDB VETERINARY DRUG NO NOT
LIMITED
92
ERYTHROMYCIN VETERINARY DRUG NO 0-0.0007
FORMIC ACID VETERINARY DRUG YES 0-3
LYNCOMYCIN
HYDROCHLORIDE
VETERINARY DRUG 0-0.3
MALATHION PESTICIDE NO 0-0.3
MONENSIN VETERINARY DRUG NO 0-0.1
OXALIC ACID PESTICIDE YES
PARADICHLOROBENZE
NE
PESTICIDE NO
OXYTETRACYCLINE VETERINARY DRUG NO 0-0.003
PERMETHRIN PESTICIDE NO 0-0.01
STREPTOMYCINE VETERINARY DRUG NO 0-0.05
PROPARGITE PESTICIDE 0-0.01
SULFATHIAZOLE VETERINARY DRUG NO NO ADI
ALLOCATE
D
RIFAMPICINE VETERINARY DRUG NO
TYLOSIN TARTRATE VETERINARY DRUG YES 0-0.03
SPINOSAD PESTICIDE NO 0-0.02
TAU-FLUVALINATE PESTICIDE YES
93
THYMOLB PESTICIDE YES ACCEPTAB
LE
a. Temporary ADI withdrawn in 1980; no ADI allocated in 1990
b. Substances considered by many national authorities as generally regarded as safe
Joint FAO/WHO expert committee on food additives. Meeting (70th:2008: Geneva.
Switzerland) evaluation of certain veterinary drug residues in food 17th report of the joint
FAO/WHO report no.954.
1.10.4. Antibiotic as Residues
Antibiotics used in animal food, can affect the public health as of their secretion in
edible animal tissues in trace amount generally called residues. e.g. chloramphenicol and
oxytetracycline residues have been found more than the regulatory standard in honey [119,
120]. Several drugs directly create toxic reactions in consumers while some other is
indirectly produce hypersensitivity or allergic reaction [121]. For example β-lactam
antibiotics can cause dermatitis, cutanious eruptions, anaphylaxis and gastrointestinal
symptoms at extremely small quantity. Such drugs include the group of penicillin and
cephalosporin antibiotics [122].
Indirect and long term hazards include carcinogenicity, microbiological and
reproductive effects. In human beings microbiological effect is one of the additional health
hazards. In consumers bacterial population can produces resistance due to the consumption
of antibiotic residues along with edible tissue like eggs, meat, honey and milk. These bacteria
may subsequently cause complication to treat human infections. Some drugs can cause
cancer in human population, like nitroimidiazoles and nitrofuran. Similarly, for a prolonged
period of time certain drugs can produce teratogenic and reproductive effect at very
minute doses consumption [123].
94
Oxytetracycline (OTC) is a broad spectrum antibiotic used as growth promoter as well treats
a verity of infection in animals. Chronic exposure symptoms of oxytetracycline contain blood
changes (leucocytosis, lung blockage, granulocytes toxic granulation, liver injury) and may
also delayed blood coagulation. It damage calcium rich organs such as bones and teeth;
some time causes nasal cavities to erode. During pregnancy, mother of Infants treated with
(OTC) may develop discoloration of the teeth. Children under seven year of age may develop
a brown coloration of the teeth. Additional chronic effects of oxytetracycline consist of
asthmatic attack, wheezing and sensitivity to the sun. Toxicological studies show that (OTC)
drug is not carcinogenic [124]. Erythromycin (ERY) is efficient in the treatment of
staphylococcal infections and also useful against Gram-positive bacteria in humans and
animals. Long contact to erythromycin particularly at antimicrobial doses and also during
breast feeding has been related to an increase chance of pyloric stenosis in little infants a
situation that causes harsh nausea in the first few months of life [125].
Erythromycin caused reproductive hazard; it has chronic exposure ‘terratogenic’. In
early pregnancy women had used erythromycin the cardiac malformation were observed in
infant [126]. Enrofloxacin (ENR) a fluoroquinolone antibiotic which act by restrain of
bacterial DNA gyrase. Embryo terratogenicity and lethality of fluoroquinolone antibacterial
in rabbits and rats has been recommended [127].
Chloramphenicol (CAP) an antimicrobial bacteriostatic used in veterinary medicine
formerly be probably carcinogenic, so it’s used is objectionable for any food producing
animals as well for honey bee’s. The European Union, Canada and United State (US) have
absolutely forbidden the treatment of chloramphenicol in the manufacture of food [128].
Chloramphenicol is predictable to be a human genotoxic and carcinogenic from
studies in humans. It is toxic to liver, kidneys and blood. Lengthened exposure to
chloramphenicol can cause bone marrow toxicity. The chronic effect of chloramphenicol is
95
aplastic anemia which is idiosyncratic (unrelated to dose, unpredictable) commonly
poisonous and could most probably be produce by residues [128]. Ampicillin (AMP) is a
penicillin derivative of β-lactam antibiotic is broadly used in swine, cattle, poultry and
honeybee’s to treat infections and as drinking water additives to stop various diseases. In
antibiotic manufacturing industrial unit workers have developed eosinophilia and asthma on
inhalation of ampicillin, it can also cause hepatitis, asthmatic attack, allergic reaction
anemia, dermatitis, thrombocytopenia, eosinophilia, thrombocytopenic purpura and
leucopenia [129].
Sulfonamide antibiotics are synthetic antimicrobial agents for preventing the
treatment of various diseases of swine, cattle, poultry and honeybee’s (Figure 1.8, i) [130].
Use of large amounts of sulfonamides in animal husbandry particularly as veterinary
medicine cause to the hazardous effects on people’s health and environment [131]. These
antibiotics can produce allergic hypersensitivity effects or toxic reactions to human health.
For these reasons, the residues of sulfonamides in the food chains must keep under control
[132].
S O
O
R1
N
R2
R3
Sulfonamide
96
N
N
NH2 S
O
O
N
H
CH3
CH3
Sulfamethazine
NH2 S
O
O
NH
O
CH3
Sulfacetamide
NH2
S
O
O
NH N
S
Sulfathiazole
Figure 1.8,i: Sulfonamides structures [132, 133].
1.10.5. Metabolites of Antibiotics
The intermediate products of metabolism are called metabolites. The term
metabolite is usually restricted to small molecule. Metabolites have various functions,
structure, including fuel, signaling, inhibitory and stimulatory effects on enzymes, catalytic
activity of their own usually as a cofactor to an enzyme, defense and interaction with other
organisms e.g. (pigments, odorants and pheromones) [133].
97
1.10.5.1. Types of Metabolites
Primary metabolite is directly involved in normal development “growth” and
reproduction. It usually performs a physiological or intrinsic function in the organism. A
primary metabolite is normally present in many organisms or cell [134]. It is also referred to
as a central metabolite which has in even more restricted meaning, present in any
separately growing cell or organism [135]. C2H4 is an example of a primary metabolites
generated in huge amount by industrial microbiology [136].
Secondary metabolites are organic compound that are not directly concerned in the regular
development, growth or reproduction of an organism [136]. Unlike primary metabolite,
absence of secondary metabolites does not result in immediate death, but rather in long
term impairment of the organism’s fecundity, survivability and aesthetics or perhaps in no
considerable change at all. Secondary metabolites are often playing vital role in plant
protection against herbivore and other interspecies. Human being uses secondary
metabolites as medicine, flavoring and recreational drugs [137].
1.10.5.2. Antibiotic Metabolites in Honey
Nitrofurans are a class of drugs, typically used as antibiotics or antimicrobial. The
defining structural component is a furan ring with a Nitro group (Figure 1.8, j) [138].
O
N
ON+
O-
O
N
O
Furazolidon
98
O
N
O
NH2
AOZ= 3-amino-2-oxazolidinone
N
O
O
N+
O-
O
N
O
N
Furaltadon
N
O
O
N
O
NH2
AMOZ = 3-amino-5-morpholino-methyl-1, 3-oxa- zolidinone
O
N+
O-
O
N
NHN
O
O
99
Nitrofurantoine
O
N
O
NH2
O
AHD = 1-aminohydantoin
ON
+
O-
O
NNH O
NH2
Nitrofurazone
NH
O
NH2NH2
SEM = Semicarbazide
Figure 1.8,j: Nitrofurans antibiotics and their metabolites structures
Before to the ban of nitrofuran and furazolidone was generally used in European countries
as a valuable veterinary antibiotic, particularly in pig husbandry. Control of residues was
based on the quantity of furazolidone concentration in tissues and blood. However, studies
concerningthe metabolism and toxicity of (FZD) and other nitrofuran revealed that the
100
monitoring of residues based only on the detection of parent nitrofuran structure did not
provide sufficient data for the estimation of real tissue contamination and their health risk
[139]. Due to doubts of the carcinogenic effects on humans, European Union were banned
the use of nitro furan in livestock production [140].
Nitrofuran, particularly furaltadone (FTD), furazolidone (FZD), nitrofurazone (NFZ)
and nitrofurantone (NFT) belong to a class of synthetic broad spectrum antibiotic which all
incorporate a characteristic 5-nitrofuran ring. Nitrofuran were usually employed as feed
additives for growth promotion and chiefly used for aquiculture i.e. fish and shrimp,
livestock that is Poultry, cattle, swine and bee colonies in the therapeutic and prophylactic
treatment of protozoan and bacterial infection such as gastrointestinal entries caused by
Salmonella species and Escherichia coli. Coccidiosis black head and chick cholera [141].
Contrary to the complete ban of nitrofuran use in livestock production, the drug are
readily available for human and animal therapy, nitrofuran is used for topical application on
skin infections and infected burns, furazolidone is available for the oral treatment of
bacterial diarrhea, giardiasis and cholera [142, 143]. Nitrofuran is commonly used to treat
urinary tract infection [144]. Furazolidone has both antibacterial and antiprotozoal activity
[145]. In human medicine it was used to treat cholera, giardiasis, diarrhea and gastro-
enteritis [146]. In veterinary medicine furazolidone has been used to treat enteritis in swine
and rabbits, and also many diseases in poultry; coccidiosis, histomoniasis, sinusitis, fowl
typhoid [147].
Chemicalname,3-(5-Nitrofurfurylideneamino)-2-oxazolidone. Molecular formula C8H7N3O5
[148].
Nitrofurans have been detected not only in treated animals, but also in animal
products, including honey. The low levels of these compounds and the complexity of honey
as a matrix present challenges for the analysis of nitrofurans. In addition, nitrofurans are
101
unstable and metabolize rapidly in vivo. Any analysis method for nitrofurans, therefore,
must be able to separate and detect these metabolites [149].
1.11. Antifungal
Antifungal are used to kill or stop further growth of fungi. In medicine, they are used
as a treatment for infection such as thrush, athlete’s foot, ring warm and worked by
exploiting differences between fungal cell and mammalian cells. Without dangerous effect
on the host they kill off the fungal organism. Unlike bacteria, both humans and fungi are
eukaryotes. Thus human and fungal cell are alike at the molecular level, making it more
complicated to find a target for in antifungal drug to attack that does not also exist in the
infected organism. However, there are habitually side effects to some of these drugs. The
improper use of these drugs, their side effects can be life threatening [150].
1.11.1. Antifungal Activity of Honey
In antimicrobial activity of honey the hydrogen peroxide is major contributor, and
the concentration of this compound in various honeys result in their changeable
antimicrobial effects. Antifungal activity of honey to stop the growth of Candida krusei,
Cryptococcus neoformans and Candida albicans [151]. Antifungal activity of honey distillate
with several antimycotic preparations against Candida albicans and common found that all
the strain opposed to conventional antimycotic agent are inhabited by the active fraction of
honey distillate. But, only incomplete data are accessible on the susceptibility of
Rhodotorulas to antiseptic and antifungal agents. Honey is used for its antifungal activity
[152].
The honey from different phytogeographic regions differ in their capability to inhabit
the growth of yeast, suggested that the botanical origin play in important rule in the
influence the antifungal activity. In addition there are vast verity of components, including
102
flavonoids, phenolic acids and other bio-molecules in various honeys. Biological activity of
honey is generally ascribed to the phenolic compounds [153].
1.11.2. Antibacterial
Antibacterial is used to treat bacterial infections, as well kill or stop further growth
of bacteria. The toxicity of antibacterial is generally considered low in humans and other
animals. However prolonged use of certain antibacterial have a negative impact on health, it
can decrease the number of gut flora. After prolonged antibacterial use, consumption of
probiotics and reasonable eating can help to replace the destroyed gut flora. Stool
transplant may be considered for patient who are having difficulty recovering from
prolonged antibiotic treatment, as for recurrent clostridium difficile infection [154].
1.11.3. Antibacterial Activity of Honey
Honey has long history of use for a wide range of disease conditions, since
ancient civilization as an effective medicine [155]. The physiological property of
honey has been recognized to production of hydrogen peroxide formed by the
enzyme glucose oxidase, antioxidant content, low pH value; osmotic action and a
variety of enzymes [156]. Antimicrobial activity of honey is one of the essential
features which allows honey to be stored for a long time without becoming spoiled
[157]. In honey hydrogen peroxide is produced by glucose oxidase secreted from the
hypo pharyngeal glands of bees. The hydrogen peroxide level is proportional to
comparative level of catalase and glucose oxidase originating from pollens [158].
Antibacterial activity of honey varies up to hundred fold in strength. This activity is
mainly due to hydrogen peroxide generated enzymically. Although honey from
“leptospermum and manuka” trees has non peroxide activity which is useful in wound
dressing. The antibacterial activity of honey is extremely important for preventing hospital
103
acquired infection by allowing the optimum wet healing conditions of honey dressing. The
autolytic debridement’s obtained with the antibacterial activity eliminate the bacterial load.
It can be obtained without risk of bacterial growth [159].
Honey can prevent wound healing or cause it to deteriorate by stimulating an
inflammatory response. Inflammation gives rise to proteolytic activity. The proteolytic
activity digests the wound bed matrixes and growth factor which are essential for tissue
repair and wound healing [160]. Honey has also a potential of direct anti-inflammation
activity in case where swelling is not due to infection. Honey supply relevant nitrification of
these cells as well as those of phagocytes. It speed up healing by exciting the growth of cells
which involve in tissue repairing [161].
1.12. Antioxidants
The name antioxidant is applied to any substances that extensively delay or stop
oxidation of an oxidizable substrate when present in low concentration, including all type of
molecule found within the living [162]. Natural antioxidant can be phenolic compounds such
as phenolic acids, tocopherol, flavonoids, amino acids, alkaloid, peptides, amines,
chlorophyll substances, carotenoid derivatives and ascorbic acid [163].
1.12.1. Phenolic Compounds
Polyphenols are phenolic compounds originating as secondary product from plants.
Polyphenols are flavonoids and phenolic acids (Figure 1.9, a) [164]. They have been regarded
to have effective antioxidant and radical scavenging activities, based on acting in different
mechanisms such as hydrogen donating, free radical scavenging and metal ion chelating
[165].
104
O
O OH
Flavonols
O
O
OH
OH
OH
OH
OH
Quercetin
O
O
Flavones
O
O
OH
OH
OH
OH
Kaempferol
105
O
O
Flavanones
Figure 1.9,a: Phenolic compounds structures
Flavonoids are compounds of low molecular weight that commonly occur bound to sugar
molecules and they can be categorized as flavonols (most widely distributed flavonoids,
including quercetin, kaempferol and myricetin), flavanones, flavones, anthocyanidins and
isoflavones [172]. Phenolic compounds are reported to show antiatherogenic,
anticarcinogenic, antithrombic, analgesic activities, anti-inflammatory and immune
modulating along with others and exerts these functions as antioxidants. The phenolic
compounds of honey are flavonoids and phenolic acids, which are considered the
prospective marker of the botanical origin of honey [166]. Phenolic acids are types of
aromatic acid compound. Together with in that class of substances containing phenolic ring
and organic carboxylic acids function (C1 to C6 skeleton). Phenolic acids can be found in
several plant species. Their content can be high in dried fruits. Phenolic acids are natural
phenols, namely p-hydroxy benzoic, vanallic, ferulic, caffeic, sinapinic, syringic and 3, 4-
dihydroxy benzoic acids [161].
1.12.2. Antioxidant Activity of Honey
The antioxidant activity of honey is due to the occurrence of different compounds
such as amino acids, carotenoid, phenolic compounds, vitamins, flavonoids, enzymes
peroxides, glucose oxidase, catalase, organic acids, ascorbic acid and proteins [167, 168]. In
many molecules, such as proteins , lipids and nucleic acids the free radicals caused oxidative
106
damage [169]. The phenolic compounds extensively contribute to human health by blocking
the formation of free radicals in the body. These compounds are mostly existing in propolis,
waxes and pollens [170].
Due to phenolic compounds, honey show antiulcer, regenerative, antimicrobial, anti-
inflammatory and antifungal activity among human [171]. Honey shows antidepressant
activity during intellectual, physical and emotional stress [172]. The bacteriostatic,
bacteriosidal, antioxidant, antiviral, antitumoral and anti-inflammatory properties make the
honey as a traditional medicine [173]. It’s proved to be effective in wound healing and burns
[174].
The antioxidant capacity of different honeys depends on the floral sources used by bee’s to
collect nectar, seasonal and environmental factors, as well as processing ways [175, 176].
However, the level of phenolic compounds present in honey is not always positively
proportional to its antioxidant. The explanation for its antioxidant activity may be due to the
presence of variable types of polyphenols, thereby providing variable scavenging activity
[177]. Darker honey is likely to have a higher antioxidant contents than light colored honeys
[178].
1.12.3. Properties of Phenolic Compounds of Honey
Phenolic compounds commonly found in honey include phenolic acids,
flavonoids and polyphenols. Honey is phenolic acids inclodes protocatequic acid,
phydroxibenzonic acid, chloroganic acid, caffeic acid, p-coumaric acid, vanallic acid,
benzoic acid, cinnamic acid and ellagic acid, Flavonoids present in honey are
kaempferol, naringenin, pinocembrin, apigenin, galangin, luteolin and chrysin
(Figure 1.9, b) [179].
OH
OH
OH
O
107
OH
OOH
OH OH
Gallic acid Caffeic acid
Ellagic acid Coumaric acid
Figure 1.9,b: Phenolic acids structures
The large and complex flavonoids greatly contribute to honey color, flavor, anti-
fungal and antibacterial activity [180]. The antioxidant behavior of phenolics are
correlated to a number of mechanisms, such as singlet oxygen quenching, free radical-
scavenging, metal ion chelating, hydrogen-donation and acting as a substrate for
radicals such as hydroxyl and superoxide [181].
The flavor of honey is slight acidic. The acid of honey accounts for less than
0.5% of the solids. The acidity level contributes not only to the flavor, but it also
responsible for the stability of honey against different microorganisms. Various acids
have been found in honey. The gluconic acid being the chief one, it arises by the
action of an enzyme glucose oxidase from the dextrose. Other acids present in honey
are lactic, butyric, formic, oxalic, acetic, succinic, tartaric, pyruvic, maleic, citric,
glycolic, alpha ketoglutaric and pyroglutamic acids [182].
OH
OH
O
O
O
OH
OH
OH
OH
OH
OH
108
1.13. Proximate Composition of Honey
Proximate composition of honey has been reported to be much emphasised on ash
content, moisture content, acidity, total soluble solids, pH, total sugars, reducing and non-
reducing sugars, glucose fructose, diastase activity, minerals energy and microbial
characteristics in the analysis of honey [183].
Mostly, natural honey is a sticky and viscous solution with a content of carbohydrates (80-
85%) mostly fructose and glucose, water (15-17%), protein (0.1-0.4%), ash (0.2%) and minor
quantities of enzymes, vitamins and amino acids as well as other substances like phenolic
antioxidants [184]. On burning and drying of honey, small residues of ash remains, which is
the mineral content varying from 0.02 to 1 % for floral honey. Honey dew honey is richer in
mineral and due to its mineral contents is said to be less suitable for storage in the winter
[185]. Although in all honey samples the major constituents of honey are nearly same, the
particular physical property and chemical composition of natural honey different according
to the plant species on which the bee’s forage [186].
The average moisture of the honey samples from all the States in Northeastern Nigeria were found tobe
within the limit of not more than 20.0 g/100 g as prescribed by Codex Alimentarius Commission [187]. Honey
contains mainly about 95% dry weight with carbohydrates. The monosaccharide’s, fructose and glucose, are the
main sugars found in honey; these hexoses are products of the hydrolysis of sucrose. In addition to these sugars, 25
others have been detected in honey samples [188].
1.14. Phytochemicals
In Greek “phyto” mean plants, phytochemicals are chemical substances naturally
occurring in plants and many of them are now recognized to have health-promoting activity
(Figure 1.10, a) [189].
109
http://jn.nutrition.org/content/134/12/3479S/F1.large.jpg
Figure 1.10,a: Phytochemicals composition chart [190].
The term is generally used to refer to those chemicals that may have biological
consequence, for example carotenoid and flavonoids, but are not recognized as essential
nutrients. Some phytochemicals are responsible for color and other organoleptic properties
Phytochemicals
Carotenoid Phenolics Alkaloids Nitrogen –
Containing
Compounds
Organosulfure
compounds
α-carotene
β-carotene
β-cryptox-
anthin
Lutein
Zeaxanthin
Astaxanthi
n
Lycopene
Phenolic
acids
Flavonoids Stibenes Coumarins Tannins
Isothio-
cyanates
Indoles
Allylic
sulfur
compounds
Hydroxy
benzoic
acid
Hydroxy
cinnamic
acids
Flavonols Flavones Flavanols Flavanones
Anthocy
-anidis
Isoflavo-
noids
Gallic
Protocat
-echuic
Vannilic
Syringic
p- Coumaric
Caffeic
Ferulic
Sanipac
Quercetin
Kaempferol
Myricetin
Galangin
Fisetin
Apigenin
Chrysin
Luteolin
Catechin
Epicatechin
Epigallocatechin
Epicatechingillate
Epigallocatechin
gillate
Eriodictyol
Hesperitin
Naringenin
Cyanidin
Pelargonidin
Delphinidin
Penoidin
Malvidin
Genistein
Diadzein
Glycitein
Formono
netin
110
such as the smell of garlic and deep purple of blueberries. Phytochemicals based dietary
supplements can also be purchased. But according to the “American Cancer Society”,
accessible scientific evidence does not support claims that taking phytochemicals
supplements is a good for long term health as consuming the vegetables, fruits, grains and
beans from which they are taken [191].
1.14.1. Phytochemicals Components in Honey
Honeys contain phytochemicals such as alkaloids, flavonoids, tannins, saponins,
terpenoids, glycoside, phlobataninsand fluoride [192]. Constitute a major group of
compounds that act as antioxidants. They consist of high oxidation reduction potentials
which allow them to act as hydrogen donors, single oxygen quenchers and reducing agent.
Delocalization of electrons over the phenolic ring and stabilization by resonance effect of the
aromatic nucleus makes the antioxidant radical uncreative (Figure 1.10, b) [193].
NH2
O
O
CH3
CH3OCH3
Alkaloids
8
7
9
6
10
5
2
3
O1
4
11
14
13
15
12
16
111
Flavonoids
OH
OH
OH
O OH
Tannins
O
CH3OCH3
OCH3
OO
OO
OO
O
O
CH3
CH3CH3
O
OH
OCH3
OCH3
OCH3
O
CH3
OCH3
OCH3 O
CH3O
O
CH3CH3
Phlobatanins
112
CH3
CH3
CH3
OH
CH3
CH3
OHCH2
Terpenoids
OOH
OH
OH O
CH3
OH
CH3
CH3
CH3
CH3
O
OH
O
O
OHOH
O
OH
OHOH
OH
O
Saponins
O
CH3
OH
CH3
CH3
OH O
CH3
Glycoside
Figure 1.10,b: Phytochemicals structures
113
1.15. Carbohydrate
Carbohydrates are hydrates of carbon, it is a large biological or macromolecule
containing of carbon, hydrogen and oxygen atoms, like water, oxygen and hydrogen atom
ratio of 1:2, the empirical formula Cm (H2O) n anywhere m could be different from n. Some
exception exists, for example Deoxy ribose a sugar constituent of Deoxy ribonucleic acid
(DNA) has the empirical formula C5H10O4. Structurally it is more precise to view them as poly
hydroxy ketones and aldehydes (Figure 1.11, a) [194].
Figure 1.11,a: Carbohydrates chart
The carbohydrates (saccharides) are divided into three chemical groups, mono saccharides,
disaccharides and polysaccharides. Carbohydrates play important roles in living organisms.
Polysaccharides serve for the storage of energy e.g. glycogen and starch , also saccharides
and their derivatives include many other imperative bio-molecules that performed key role
in the fertilization, immune system, development, blood clotting and preventing
pathogenesis [195].
Carbohydrates
Glucose
Fructose
Glactose
Maltose
Sucrose
Lactose
Starch
Cellulose
Glycogen
Monosaccharide Disaccharide Polysaccharide
114
O
H OH
OH OH
OH
OHH
H
β-D-glucose
O
OH OH
H H
OH H
H OH
H
OH
β-D-fructose
OOH
H
OH
OH
OHH
H
O
OHO
H H
OH H
H OH
H
OH
α-D-sucrose
Figure 1.11,b: Carbohydrates structures
1.15.1. Carbohydrates in Honey
Carbohydrates are the main constituent of honey produced by honey bees from
nectar source, which is transform through the action of several enzyme, mostly β-
fructosidase , α, β- amylase and α, β -glucosidase [14, 196].
115
More than 95% of the honey solids are carbohydrates. About 22 sugars have been
found in honey but dextrose and laevulose are the major sugars. Majority of these sugars
are more complex then laevulose and dextrose (monosaccharides). Ten disaccharides have
been identified includes isomaltose, nigerose, turanose, maltolose,
OH
OH H
H OH
OH
α-D-turanose
O
OH
OH
OH
OH
O
OH
OHOH
OOH
β-Kejobiose
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O
OH
OH
OH OH
O OOH
OH
OH
OH
α-Palatinose
Figure 1.11,c: Carbohydrates structures
sucrose, turanose, gentiobiose, laminaribose, β-trehalose, maltose and kojebiose. Tri
saccharides are also found consist of maltotriose, erlose, melezitose, centose 3-a-
5isomaltosylglucose, l-kestose, isomaltotriose, panose, isopanose and theanderose. All these
sugars are present in very small quantity (Figure 1.11, b) [197].
The presence of oligosaccharides (melezitose, rifinose and erolose),
monosaccharide’s (glucose, fructose) and disaccharides (sucrose, maltose, kejobiose,
isomaltose, turanose) in large quantities produce and on other hand also in very specific
honey is documented [198].
Disaccharides of honey are mainly constituted by regioisomers of a-glycosyle
fructose and α-glycosyle glucose; disaccharides with α-glycosidic linkage are present in small
amount while fructosyle-fructoses are very scarce. The more abundant trisaccharides are
derivatives of sucrose (Figure 1.11, c) [199, 200].
Generally, carbohydrates are one of the most important components in many food
items and they may be either present as isolated form or associated form to other
macromolecules [201]. Sugars are simple carbohydrates and are important for everyday life
biological functions such as providing energy for running vital roles of the living body [202].
117
The majority of the natural sugars contain 6 or 12 carbon atoms in their molecules. Sugars
are crystalline, soluble in water and generally have a sweet taste. The commercial sugar is
the disaccharide sucrose white sugar. Usually, fructose is slightly sweeter than sucrose and
glucose is less sweet [203]. The sweetness of mono-floral honey a honey made from a single
flower source is dependent on the ratio of fructose to glucose that results from the bee’s
processing the nectar of the homo mono-specific flower. Most of the honey sold in the
markets is a blend of varieties, to create a consistent flavor and sweetness profile. However,
most of the honey’s fructose becomes predominating, thus, it achieves creation of a sweet
honey taste [204].
1.16. Contamination in Honey
Heavy metals are chemical elements that are at least five time the specific gravity of
water. At (4oC) the specific gravity of water is 1, just confirmed, specific gravity is quantified
of density of particular amount of a solid material when it is compared to an equal quantity
of water. A few identified toxic metallic elements with a specific gravity that is five or more
than that of water are lead (11.34), iron (7.9), mercury (13.546), cadmium (8.65) and arsenic
(5.7) (Figure 1.12, a) [205].
1.16.1. Non Toxic Heavy Metals
For a healthy life certain heavy metals are nutritionally necessary in small quantities.
Some of these are referred as the trace elements e.g. Manganese, iron, zinc and copper. In
foodstuff, vegetables and fruits these elements or some form of them are found naturally
and also available in multivitamin products commercially [206]. Diagnostic medical
application direct injection of gallium include during radiological procedures. Lead used as a
radiation shield around x-ray equipment, also dosing with chromium in parenteral nutrition
mixtures [207].
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1.16.2. Toxic Heavy Metals
When heavy metals are not metabolized and accumulated in body soft tissues they
become toxic. It can penetrate to human body from air, water, food or absorption through
skin when they come to contact with humans in industrial, agriculture, manufacturing or
residential sitting. Ingestion is the main ordinary path of exposer in children and industrial
depiction accounts for a common rout of exposer for adults
[208].
Figure 1.12,a: Toxic heavy metals
Children may develop toxic levels from the normal hand to mouth activity by
actually eating object like paint, dirt or chips otherwise who come in contact with
119
contaminated soil [209]. Priority list for 2001 called the “Top 20 hazardous substances “has
complied by ATSDR with corporations of United State Environmental Protection Agency. The
heavy metals cadmium, lead, mercury, arsenic, iron and aluminum, as noted earlier there
are 35 metals are concern, 23 of them are called heavy metals. These metals cause toxicity.
This protocol will address the metals that are most expected come across in our everyday
surroundings. Briefly covers with the heavy toxic metals that are incorporated in the ATSDR,
s list [210].
1.16.3. Heavy Metals in Honey
Beside the usefulness of honey, there are certain problems like heavy metals and
pesticides contaminants exist in honey make them infected. Bee’s transport these
contaminants to beehives from plants, soil and water [211]. In honey, minerals content is
about 0.17%; however it changes within a wide range. For environmental pollution, honey
has been considered is a biological indicator because honey bee’s create bioaccumulation
process. Consequently in honey the heavy metals concentration represents their quantity in
the whole regions, as the forage area of the hives is extremely large and the bees come in
contact not only with air and soil but also with water [212]. Lead and cadmium are the most
toxic heavy metals. These originates mostly from metals industries, traffic vehicles,
incinerators is transported from the soil to plants and also through air can directly polluted
honeydew and nectar [213]. In honey quality controlled and nutritional aspects is based on
heavy metals. Supposed or known metal toxicity are objectionable so that in some countries
a limit set for lead is 1 milligram per kilogram [214].
In small sufficient quantities metals are important for all life forms. It penetrates into
the cell like cations but their insertion is strictly regulated because all metals are toxic in
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large amount [215]. Like other vertebrates the humans being require metals cations,
because they comfort the improvement of many processes of critical significance. The
division of the metals in neutral, toxic and essential, may be misled and often false, because
all the essential elements in small quantity become toxic and more toxic in large doses [216].
Honey is a dietary supplement and show therapeutic values due to its traces level of
important minerals that are necessary for health [217]. Metals intakes in traces quantity are
important in daily diets due to their essential nutritional value. The traces minerals such as
zinc, iron, manganese and copper are important part of biological system [218]. For human,
food is one of the main sources and diet is the main rout of exposure to trace metals.
Therefore, to asses risk to human health for these elements, analysis of food samples and
collecting information about dietary intake is also important [219]. Honeybee’s may
constantly expose to contaminants during the foraging activities in the areas surrounding
the apiary [220]. Bee’s and their products can serve as bio-indicators for contamination as
they fly intensively in the area about 3 kilometers [221].
1.17. Mycotoxin
Mycotoxin is derived from two Greek words “mukos”, mean “fungus” and toxikon”
mean “poison”. So it is a poisonous secondary metabolites formed by organisms of the fungi
kingdom, frequently known as molds [222]. Mycotoxins naturally produce due to the fungal
growth on some food material such as fruits, nuts and spices. Aflatoxins is the most
commonly observed mycotoxin having types as B1, B2, G1, G2 and ochratoxin A. in many
developing countries have been shown that aflatoxin directly damage DNA and cause cancer
of liver in laboratory..The term mycotoxin is usually kept for the toxic chemical products
produce by fungi that readily occupied crops [223]. Single mold species may produce many
different mycotoxins as well the several species produce same mycotoxin [224].
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The majorities of the fungi are use oxygen (aerobic) and almost found everywhere in very
small quantities due to the minute’s spore’s size. They consumed organic matter wherever
humidity and temperature are sufficient. In favorable condition, fungi proliferate into
colonies and mycotoxin level become high. The cause for the production of mycotoxin is not
yet identified, they are not necessary for the development or growth of the fungi [225].
Some of the health effects found in humans and animals include death, health problems or
identifiable diseases, irritants or allergies and weaken immune system without specificity of
a toxin. Some mycotoxins are harmful to other microorganisms such as bacteria and fungi,
penicillin is best example [226].
Mycotoxin exposure can produce both acute and chronic toxicities ranging from
death to poisonous effect upon the alimentary tract, pulmonary, cardiovascular and central
nervous system. Mycotoxin may also mutagenic, immunosuppressive, carcinogenic and
teratogenic [224]. The ability of some mycotoxin to compromise the immune response and
hence to reduce resistance to infections disease is now extensively considered to be the
most important effect of mycotoxin [227]. Mostly in developing countries i.e. Canada and
United State of America, arising from the shock of mycotoxin on the livestock and feed
industries are of the order of five billion dollar’s food staples e.g. groundnuts and maize are
disposed to contamination, it is likely that significant additional losses will occur amongst the
human population because of morbidity and free mature death related with the expenditure
of mycotoxin [228].
1.17.1. Aflatoxins
Aflatoxins are a type of mycotoxin produced by Aspergillus species of fungi such as
Aspergillus parasiticus and Aspergillus flavus. The umbrella term aflatoxins refers to four
different type of mycotoxins produced which are (B1, B2, G1and G2) respectively (Figure
1.12, b) [229]. The most toxic and potent carcinogenic, aflatoxins B1 has been directly
122
interrelated to adverse health effects, such as liver cancer in various animal species. Also, in
particular environmental condition aflatoxins B1 can permeate through skin dermal
exposure can cause serious health risks [230].
O
O
O
O
O
CH3
O
Aflatoxins B1 Aflatoxins B2
O
O
O
O
O
CH3
O
O
Aflatoxins G1 Aflatoxins G2
Figure 1.12,b: Aflatoxins B1, B2, G1 and G2 [231].
Aflatoxins are basically associated with commodities produced in tropical and sub-
tropical such as spices, cotton, pistachios, peanuts and maize. Subclinical exposure does not
lead to symptoms as chronic or acute aflatoxicosis. Particularly children’s are affected by
aflatoxins exposure, which leads hindrance development and stunted growth [232].
Aflatoxin B1 exposure can cause immune suppression and increase viral load in human
immune deficient virus (HIV) positive individuals [233]. Medical research specify that a
O
O
O
O
O
CH3
O
O
O
O
O
O
CH3
O
O
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regular diet including apiaceous vegetables such as parsley, carrots, celery and parsnips may
decrease the carcinogenic effects of aflatoxins [234].
Aflatoxins are considered is the most problematic mycotoxin, their expression
related disease is influenced by factor such as age, sex, species nutrition and the possibility
of current exposure to other toxins. The aflatoxicosis is primarily a hepatic disease because,
in mammalian main targeted organ are liver [235]. In humans, conditions increased the
likelihood of aflatoxicosis include environmental condition, limited availability of food that
favor mold growth on foodstuffs and lack of regulatory system for aflatoxins control and
monitoring [236].
1.17.2. Aflatoxins in Honey
In honey several changes produced during storage, the most significance change
occur in honey is spontaneous fermentation caused by osmophalic yeast [237]. Yeast, spore
and mould producing bacteria are the microbes of usually concern in honey. These
microorganisms may take part in several activities such as spoilage of provision, metabolic
conversion of provision, production of enzymes ,antibiotics, growth factors, (vitamin and
amino acids), inhibition of competing microorganism and mycotoxins. Marketable honey
distribution can be presented in large quantity and also package for retail sale.
Microbiological uniqueness of honey are inheriting to safety and quality [238].
Beside the financial loss due to food contamination, along with mycotoxin, aflatoxins
could be more harmful for human health, they are mutagenic, teratogenic, carcinogenic and
toxigenic [239]. In preliminary study on honey in Portugal, reported less contamination with
fungi such as penicillium, yeast, mucor species and many species of Aspergillus genus,
particularly Aspergillus flavus, Aspergillus fumigates and Aspergillus candidus. From these
potentially pathogenic species inclined patients can get harm. In clinical form of botulism,
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bacterial spores grow and produce toxins in the intestinal tract of exaggerated infants less
than one year of age [240].
1.18. Aims and Objectives of the Present Study
To evaluate the antibiotic residues and their metabolites in honey
To evaluate the quality parameters such as phenolic, antifungal, antibacterial
and antioxidant activities of both natural and farms honey
To evaluate proximate and phytochemicals composition of honey
To evaluate the contaminants such as heavy metals and aflatoxins in honey.
1.19. Suggestion for Further Work
Methods can be developed for degradation of antibiotic residues in honey by
thermal, electrical conduction or by X-rays
Value of temperature, current and radiation can be determined which degrade the
residues and not disturbed the quality and composition of honey
Amount of antibiotics for bees can be detected to combat bee’s diseases
Checking effects of packing materials which is used for the storage of honey
Study effects of X-rays on honey, beekeepers passed X-rays from honey for
uncrystalization purposes
Evaluation of amino acids profile in natural and farm honey of Pakistan
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CHAPTER-2
2. LITRATURE AND REVIEW
Antibiotics residue in honey have recently become a main customer concern. It has
become evident that antibiotic residue in honey initiate habitually not from the environment
but from improper beekeeping practices. According to Bagnadov et al., (2006) in the
European Union, treatment of Amirican and Europian brood diseases of honey bee’s with
antibiotics are not acceptable, while in many other countries they are extensively used.
Consequently, there are no Maximum Residues Limit (MRL) levels for antibiotics in most
European Union countries, which mean that honey containing antibiotics residues are not
allowed to be sold. As no residues are allowable, no maximum residue limit is recognized.
However some countries like Belgium, United kingdom and Switzerland have established
action limits, which usually lie between 0.01 to 0.05mg/kg for each antibiotic group [241].
Reybroeck et al., (2003) reported during 2000 - 2001 that the honey local samples
were checked for the presence of veterinary drug residues. Sulfonamides in three out of 72
samples, streptomycin was found in four out of 248, tetracycline in two out of 72.
Antibiotics residues of chloramphenicol and β-lactam were not detected. Streptomycin in
fifty one out of 102 samples, chloramphenicol 40 out of 85, sulfonamides in 31 out of 98
samples, tetracycline in 29 out of 98 samples was detected in imported honey samples. For
the tetracycline and streptomycine contamination, the majority cases involved the
beekeepers admitting to having added foreign honey to this production [242].
Ortelli et al., (2004) reported that chloramphenicol concentration measured in
honey between 0.4 and 0.6 µg/kg, with 6 sample containing approximately 0.8-0.9 µg/kg
and 2 containing approximately 5 µg/kg (just below the Swiss limit) [120].
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Saridaki-Papakonstadinou et al., (2006) reported that 251 honey samples were analyzed in
Greece to detect derived residues of tetracycline by liquid chromatography. Tetracyclines
were detected in 29% samples. Majority of the honey samples contained 0.018 to
0.055mg/kg residues while some others had residues in excess of 0.100 mg/kg [119]. Centre
for Food Safety (CFS) (2006) reported that 2 of 19 samples of honey collected for
examination of chloramphenicol antibiotic contained trace quantity in one brand in honey
produced in Jiangxi. Another brand produced in Zhuhai in traces amount other antibiotics
detected in honey samples are sulfamethoxazole, ciprofloxacin and streptomycine can
normally be used in food of animals [243].
Gunes et al., (2008) reported that erythromycin residue concentration in honey
samples ranging from 50-1776µg/kg, and also found that the contamination of erythromycin
were 8% in honey samples [244]. Vidal et al., (2009) reported that the presence of
erythromycin were 8.6µg/kg in 3 out of 16 honey samples of Almeria and Granada [245].
According to the Solomon et al., (2006) honey and nectar samples showed ampicillin 2 - 29
and 3 - 44µg/kg, kanamycin 17-34 and 26-48µg/kg and streptomycin 4-17 and 11-29µg/kg
respectively [246].
According to Mahmoudi et al., (2007) 3855 samples of honey were tested 1.7%
samples were non complaint, in European Union standard antibiotic were found in honey
samples in the range sulfonamides 5-4592 µg/kg, streptomycin 3-10,820 µg/kg,
tetracyclines5-2,076 µg/kg, nitrofuran 0.3-24.7 µg/kg, chloramphenicol 0.1-196µg/kg,
quinolones 1-504µg/kg and tylosine 2-18µg/kg [115].
Gunes et al., (2008) reported that samples of four honeys were contaminated with
erythromycin residues at concentration ranging from 50-1766ngg-1. An erythromycin
equipped cake feeding assay was also performed in a defined hive to test the transfer of
erythromycin residues to the honey matrix, after 3 month dosing the residues level in honey
127
was just about 28nano grams [244]. Thompson et al., (2005) reported that oxytetracycline
(OTC) in liquid form have very high residues level in honey after 8 weeks application residues
range 3.7 mg/kg were found [247]. Reybroeck et al.,(2003) reported that the detection of
chloramphenicol in both the imported honey samples were at the level of 3.6-3.7µg/kg in
Capilano,s honey (Australia) [242]. Vidal et al.,(2009) reported that commercial honey
samples contained 8.6µg/kg while honey from one bee farm contained traces amount
residues of sulfadimidin, tylosine, sulfachlorpyridazine and sarafloxacin [245].
Zhou et al., (2009) reported that total of 57 real royal jelly samples collected from
supermarkets and beekeepers were examine for seven fluoroquinolones used in
beekeeping, verses norfloxacin, ciprofloxacin, pefloxcin, ofloxacin, enrofloxacin, defloxacin
and danofloxacin were analyzed by high performance liquid chromatography with
florescence detection. Norfloxacin , ciprofloxacin and ofloxacin were detected in
concentration ranging from 11.9 -55.6ng/g in some royal jelly samples while defloxacin was
detected at concentration of about 46.8ng/g in one sample through it is infrequently used in
beekeeping [248]. Baggio et al., (2004) reported that the Italian honey samples were tested,
contained 2-7% tetracycline and sulfonamide [249].
According to Verzegnassi et al., (2003) showed great part of Chinese honey and
result from different laboratories, but honey from various countries also contain bigger
quantities of chloramphenicol then the European Union MRL of 0.3µg/kg [250].
Nitrofurans are broad spectrum antibiotics used with bacterial infections to treat animals as
well bees. As a result of dosing bees with these, antibiotics and their metabolites are
sometime found in honey. Nagrin et al., (2013) reported that nitrofurone were usually used
as feed additives for growth endorsement and mainly used for aquiculture like fish, shrimp,
livestock, cattle, poultry, swine, bee colonies for prophylactic, therapeutic treatment of
protozoan and bacterial infections such as gastrointestinal entries caused by salmonella
128
species and Escherichia coli. Due to concern about the drug residues, their carcinogenicity
and possible harmful effects on human health [251]. Kleinschmidt et al., (2010) reported
that Nitrofuran metabolites AOZ, SC were also found in honey [252].
According to European Union (2010) metabolite of the veterinary antibiotic
nitrofurazone which is genotoxic to humans and as such in Europe, is a prohibited substance
for all food-producing animals under Commission Regulation No 37/2010. SEM is
consequently used as an indicator in tests to detect the nitrofurazone in food producing
animals. In European Union; SEM in food should not exceed the (MRPL) of 1µg/kg as defined
in Annex II of Commission Decision 2002/657/EC. Semicarbazoid (SEM) as a reliable marker
of nitrofurazone use in honey production needs to be addressed in order to protect
consumer safety and confidence in the product [253].
According to Food StandardAgency Scotland (FSAS), a consultant analytical
laboratory with UKAS accreditation analyzed 13 honey samples in 2010 for the nitrofuran
metabolites 3- amino -2- oxazolidone (AOZ) 3-amino-5-morpholinomethyl-2-
oxazolidone(AMOZ) and semicarbazide (SEM).1-aminohydantoin (AHD) by High Performance
Liquid Chromatography/ mass spectrometric method (LC-MS) [254]. Two samples were also
subjected to pollen analysis of semicarbazide (SEM), but no other metabolites were
detected in several samples. The 2010 crop of Scottish heather honey had been found to
contain SEM, with confidence that it had not arisen from use of nitrofuran. Dozens of
samples from different beekeepers and many different apiary locations had been tested,
with all heather honey samples showing the presence of SEM, at consistent levels between
0.6 and 1.8μg/kg. Samples of other honeys, produced from the same hives but at different
times and or locations, did not show SEM, suggesting a link between SEM and heather honey
In 2010 SEM has also been reported in a sample of honey collected in England, and SEM has
recently been reported in two samples of wild forest honey (Kamahi) honey imported to the
129
UK from New Zealand, one of which has been reported formally directorate of veterinary
medicine (VMD 2012) [255]. Reybroeck et al., (2010) reported that approximately 20
honeys from each of Belgium, Italy, Portugal, Spain, Switzerland, and UK were tested in 2009
for the presence of nitrofuran metabolites (SEM, AMOZ, AOZ, and AHD), and all were found
to be negative [256].
According to European commission 2002 meets the criteria set out by the EU for the
unequivocal confirmation of the presence of these metabolites at concentrations well below
1.0 µg/kg [257]. Mccalla et al., (1983) reported about the drug residues their carcinogenicity
and potential harmful effects on human health [258]. According to commission decision
(2003) countries with products intended for the European Union are bound by the same
regulation as locally produced food [259]. Ahmed et al., (2008) reported that in mammalian
cell the formation of mutagens and toxicity in vitro is less understood. When cell were
exposed to furazolidone the irreversible damage of DNA of human epithelial cell as well
hormone disorder (reflecting endocrine dysfunction) occur usually [260].
Vass et al., (2008) reported that during 2002- 2003 the global nitrofuran crisis
exposed frequent finding of tissues bound residues in poultry and aquiculture products
imported to European Union countries from Taiwan, Thailand, China, Vietnam and Brazil
[140]. O’Keefe et al., (2004) reported that nitrofuran residues were also establish in poultry
and pork muscle produced in European countries such as Bulgaria, Romania, Italy, Greece
and Portugal [261]. According to the European Commission (2008), nitrofuran contamination
in product originating from over nine countries in 2007, maximum occurrence being from
china (37%), India (37%), Bangladesh (10%) and Thailand (5%) in a verity of products
including honey, meat and shrimp [262]. It also reported by European commission (2008)
that the aquiculture products from Asian countries are often contaminated by SEM and AOZ.
In frozen peeled black tiger shrimps from India, highest concentration of AOZ was 150µg/kg
130
while nitrofuran were found at lower concentration 10 to 63µg/kg also not rare [262].
Guerrini et al., (2009) reported that honey of stingless bees act as a protective agent against
DNA damage and could present interesting evidence in relation to the determined
antioxidant competence [263].
According to Kilicoglu et al., (2008) reported the effect of honey on apoptosis and
oxidative stress in investigational obstructive jaundice and establish that honey reduce the
harmful effects of bile duct ligation on the hepatic ultra-structure. This effect may be due to
its anti-inflammatory and antioxidant activities [264]. According to Korkmazet al., (2009)
that in the body N-ethylemaleimide is a sulfhydryl blocker which impairs sulfhydryl
dependent antioxidant system. The result involve that depletion of glutathione
concentration play an informal role in NEM-induce liver injury and this hepatic protective
effect of honey may be mediated through sulfhydryl sensitive process [265].
Zaid et al., (2010) concluded that honey could be an alternative to hormone
replacement therapy. The honey had positive effects on menopausal rats by preventing
uterine atrophy, suppression of increase body weight and increase bone density [266]. Chun
et al., (2005) reported that honey is a source of antioxidants, but in honey the content of
polyphenols is not very high as compare to some vegetables and fruits for example
strawberries contain 2250.0µg/g polyphenols, asparagus 641.5µg/g, apple 1183.0µg/g and
plums 3686.6µg/g, because of dominant concentration of carbohydrates (fructose and
glucose) in honey. Some honey have high concentration of phenolic compounds like fruits
and vegetables for example heather and buckwheat honey had a higher content of phenolic
compounds as compared to mushrooms 112.5µg/g, honey melon 114.5µg/g and carrots
84.0µg/g respectively [267]. Al-Mamary et al., (2002) reported that the antioxidant activity
of honey can be related to the high concentration of phenolic compounds were (0.05 to
125.17mg/l) in comparison with other types of honey reported in literature [268]. Yao et al.,
131
(2003) reported that the compound present in chromatogram that have similar phenolic
acid and flavonoids spectra and chromatographic behavior but have not identified due to
lack of accessibility of standard compounds. These phenolic compounds were already
reported in honey [269].
According to Aljadi et al., (2003) the total phenolic content extract of the two honey
samples used for the antioxidant and antibacterial properties study, calculated by weight
before the dissolution with (DMSO), was in average 13.0 mg/100g and 4.1 mg/100g for dark
and clear honey [270]. Kamaruddin et al., (2004) reported that the darker honey was higher
antioxidant activity then that of clear honey, which was due to the difference in their
phenolic compounds contents and consequently their floral source, as well verified by Al-
Mamary et al., (2002). The results obtained shows that all tested samples were
antioxidatively active, their RSA varying between 47, 84 and 62, 99 % Inhibition of the DPPH
solution [157].
Akbulutet al., (2009) reported that honey of different verities from various geographical
regions and countries have been shown high antioxidant properties. Turkish honey (red
pine) produced by marchalina hellenica have successfully DPPH scavenging, indicative of its
antiradical activities [271]. Al-Hindi et al., (2011) reported that samples of Saudi Arabian
honey were confirmed to show antioxidant activities [272]. Rodríguez et al., (2009) also
reported similar antioxidant properties for Peruvian honey [273].
Oddo et al., (2008) was reported that Australian honey show high antioxidant properties,
produce by the stingless bees trigona carbonaria [274]. Kishore et al., (2011) reported that
Malaysian tulang honey has been shown good antiradical and antioxidant activities,
produced by giant Asian bees Apis dorsata [275]. Van den Berg et al., (2008) reported that
antioxidant activity have also been documented for American buckwheat [276]. Eraslan et
al., (2010) also reported that in honey the main phenolic compounds and flavonoids includes
132
syringic acid, elligic acid, ferulic acid, chlorogenic acid, cinimic acid, caffic acid, gallic acid,
benzoic acid, coumaric acid, kaempferol, quercetin, myricetin, isoramnetin, galangin,
chrysin, hesperetin and luteolin [277].
Petrus et al., (2011) reported that in most honey samples some of these bioactive
compound such as kaempferol, luteolin,quercetin,alangin and isorhamnetin are found, while
in few honey verities naringenin and hesperetin are found [278]. Khalil et al., (2011)
reported that catechin was found to be the most common flavonoids investigated in
Malaysian honey [279]. According to Gheldof et al., (2002) about fourteen phenolic
compounds were identified the ten phenolic acids and four flavonoids and the phenolic
pattern of honey contained protocatequic acid, gallic acid, p-hydroxybenzoic acid, p-
methoxybenzoic acids, syringic acid, vanillic acid, sinapic acid, coumaric acid, p-
methoxycinimic acid and cinnamic acid, as well as the flavonoids rutin, isoquercetin, morin
and quercetin. Some of these phenolic compounds were already identified by AL, M et al.,
(2009) in other honeys [280, 281]. Maria et al, observed that the antifungal activity of honey
stop the growth of Cryptococcus neoformans, Candida krusei, and Candida albicans [151].
Obaseik Ebor et al., (1984) evaluate the antifungal activity of honey distillate, some
antimycotic preparations against Candida albicans and observed that all the strained
opposed to predictable antimycotic agents are occupied by the active fraction of honey
distillate [282].
Wahdan et al., (1998) reported that 21 types of bacteria, including Staphylococcus sp,
Escherichia coli, Pseudomonas species, Klebsiella species, and two types of fungi in vitro,
honey neutralized more pathogens than sugar control, and undiluted honey completely
inhibited the growth of all 21 bacteria [283]. Taormina et al., (2001) recognized the effect of
honey on gram negative bacteria is due to the existence of powerful antioxidants and
133
hydrogen peroxide as also natural pH, which is incompatible for bacterial development and
to the presence of flavonoids, lysozyme and phenolic acids [284].
According to Chauhan et al., (2010) the most susceptible bacteria include P.aeruginosa and
E.coli with ZDI of honey for the isolate range 6.94 to 35.95mm and MIC in the range of 0.625
to 5.0 mg/ml, respectively [285]. Al-Namma et al., (2009) reported that honey has
potential and therapeutic properties at also better inhibitory effect on Gram negative
bacteria E.coli, P.aeruginosa, and S.typhi are more disposed then opted test organism [286].
Wilkinson et al., (2005) observed the activity of thirteen honeys at four concentrations (1,
2.5, 5 and 10% v/v) with matching dilution of an artificial honey (a solution containing
principal’s sugars found honey) and using against the test organisams as P.aeruginosa and
E.coli [287].
Packer et al., (2012) as well Blair et al., (2009) both reported that proteome and
transcriptome studies on bacteria how respond to treatment a unique multimodal mode of
action have found in honey [288, 289]. Theunissen et al., (2001) reported that lists 64
different bacterial and 13 fungal species on which antimicrobial action has been tested
[290]. Irish et al., (2006) reported that honey is a natural product that used for its antifungal
activity [291]. Bouleraa et al., (2008) reported that honey has a efficient antibacterial activity
and very useful clearing infection in wound as well protecting them from become infected
[292].
According to Kačániová and Kňazovická et al., (2009) that honey and propolis has been
found to have antimicrobial activity and has also attributes to specific chemicals in the
honey and propolis [293]. Miroslava kacaniovaet al., (2009) reported that honey samples
showed antifungal activity with 25% concentration against fungi Candida parapsilosis,
Candida glabrata, Candida tropicalis, Candida crusei and Candida albicans strains are
presented by the antifungal antibacterial activity of honey samples were assessed by the
134
(mm) diameter of the obtained sterile zone around the disk no inhibition zones were seen
against the yeasts investigated in the 25 % concentration of honey samples [294]. According
to Muli et al., (2008) that inhibitory effect of honey samples were noted for S.typhi and
B.subtilis. While, no inhibitory effect was noted on A.niger, E.coli,C.albicans and S.aureus
[295].
Mohammed et al., (2008) reported the different concentrations of the two honey samples
had good growth inhibitory effect on the tested microorganisms [296]. Similar result was
previously reported by Al-Nahari et al., (2015) [297] for E. coli and P. aeruginosa, Agbaje et
al., (2006) for E. coli, K. pneumonia [298]. Alqurashi et al., (2013) reported that MIC
observed with Sidr honey was lower, showed by MIC assay (20 mg/ml) for the tested
microorganisms while those of mountain honey ranged from 20 to 40 mg/ml. For both
honey samples the MBC values were in the range of 20 to 40 mg/ml. The lowest MBC value
(20 mg / ml) was against A. baumannii [299]. The present findings are consistent with the
results reported by Hern et al., (2009).Comparing the mean (standard deviation) of the
inhibition diameters for tested bacteria at various concentration of honey. It was observed
that statistically significant difference in the value (p≤0.05) between microorganisms at all
the honey concentration. All the different concentrations of both honey samples (10 to 80%)
showed growth inhibitory activity against E. coli [298].
This contrasts with the result reported by Hegazi et al., (2011) reported that the different
types of Saudi honey were less inhibitory against E. coli than other bacteria. All the tested
bacteria were sensitive to Sidr and Mountain honeys at 40 to 80% concentrations. The
antibacterial activity of Sidr honey was higher than those obtained by Mountain honey. In
overall antibacterial activity variation were observed due to change in the level of hydrogen
peroxide attain, and some cases to the level of non-peroxide factor. In honey the content of
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non-peroxide factor was evidently related to the floral source and sometime accounted for
the main part of the antibacterial activity [300, 301].
Williams et al., (2009) reported that the various quality parameter of honey are very
important compositional essential for its use as medicine or as food [302]. Belitz et al.,
(2009) reported that honey contains approximately 400 compounds and alone the united
states have more than 300 unique verities of honey depending upon the floral source.
Honey mostly composed of sugars 79.6% and water 17.2% which accounts roughly. Main
sugars of honey are dextrose and laevulose which constitutes 31.28% and 38.19%
respectively; remaining is maltose 7.3% and sucrose 1.3%. Minor constituents of honey
includes protein (0.266%), acids (0.57%), amino acids (0.1%) little amount of nitrogen
(0.043%) and minerals (0.17%) [303].
According to Kaškoniene et al., (2009) that the minutes quantities of components
like phenolic compounds, pigments, vitamins, colloids, sugar alcohols, aroma substances and
flavors which all collectively account for the 2.1% of complete honey composition [304].
Kamal et al., (2002) determined that quality parameters include color, acidity, ash contents,
electrical conductivity, HMF contents, sugar contents and moisture. In different honeys the
pH was found to be significantly different from each other [305]. European Union (2002)
reported that The pH of the honey samples was observed (6.56 + 0.05) [306]. According to
Nasiruddin et al., (2006) that in local honeys the acidity range from 23.55 to 58.52meq/kg
were reported. While Kamal et al., (2002) reported that this range lies between 6.73 to
22.9meq/kg respectively [307]. According to Malika et al., (2005) in honey, Ash represents
inorganic residues which can be calculated after carbonization. The moisture content of
honey is extensively related to the level of development in the hive and harvest season.
Electrical conductivity depends upon the mineral content of the honey. These parameters
are extremely important for the shelf life of honey [308].
136
Mairaj et al., (2008) reported that the moisture content in honey ranged from 15.6
to 19.2% [309]. Agbagwa et al., (2010) reported that the ash content of the Nigerian honey
samples varied between 0.05 and 0.79% [310]. Mateo et al., (2004) reported that honey
contain about 85% sugar mostly glucose and fructose produce by the hydrolysis of sucrose.
The comparative amount of glucose and fructose is important for the unifloral honey
classification [311].
Subramanian et al., (2007) reported that Hydroxy Methyl Furfural (HMF) is formed when
fructose is decomposed and depends upon the heating temperature, storage time and pH.
The HMF is used as standard for testing honey’s freshness and overheating of the honey
[312]. EU recommends a maximum HMF concentration of 40mg/kg [313]. Gonzalez M et al.,
(2005) reported that honey composed varying quantity of minerals substances ranging from
0.02-1.03g/100g[314]. Kivimaa et al., (2014) reported the traces element contents of honey
depends mainly on the botanical origin of honey, darker honeys have higher content then
light blossom honeys e.g. heather, chestnut and honeydew [315]. According to Nozal Nalda
et al., (2005) that it was probable to discriminate between different unifloral honeys by
determination of various traces elements by measuring Al, Mn, Ca, Mg, Cd, Cr, Ni, Cu, B, Zn
and P [316]. Erbilir and Erdoğrul et al., (2005) observed that Cd and Mn levels were 0.32 and
0.03 ppm in honey samples, respectively [317]. Temamogulları et al., (2012) determined that
Mn, Pb and Cd contents in honeys were 0.32-4.56 ppm, 8.4-105 ppb and 0.9- 17.9 ppb,
respectively [318]. Yilmaz and Yavus et al., (1999) reported that the honey obtained from the
different areas of southeastern Anatolia, level of Mn was 1.0ppm [319]. According to Yarsan
et al., (2007) the Mn level of honey was 0.49ppm [320]. In another study, Fredes and
Montenegro et al., (2006) reported 0.01-0.11 mg/kg and Conti and Botre et al., (2001)
reported 3.3-45.0 μg/kg respectively [220, 321]. The mean of Cd level of honey was 0.003
ppm reported by FüsunTemamoğulları et al., (2012) [318].
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Frias et al., (2008) reported that Pb (31.50 mg/kg) honey, and Cd (46.32mg/kg) in
honey from Tenerife [322]. Seidel et al., (2008) reported that lower levels of Pb in honey
(0.98 mg/kg) than in propolis (5.74 mg/kg) from Poland [323]. In agreement with Chudzinska
and Baralkiewicz et al., (2010) who also reported Pb values lower than 1 mg/kg, with higher
contents in rape honey [324]. Sahinler Nuray et al., (2009) reported that in Turkish honey
the highest Pb level exists in cotton honey, 1.29 mg/kg [325]. Kacainova et al., (2009)
reported that in honey from Slovakia, Pb is lower than 0.001 mg/kg, below their detectable
limit [293].
The level of metals were below the tolerable amount prescribed by the Czech Bylaw
(298/1997) Pb - 8000μg/kg and Hg - 500μg/kg [324]. The concentrations of Pb and Hg varied
in individual groups of honey. In a Polish study, Madras-Majevska et al., (2002) reported that
mercury (Hg) were calculated in different bee product were studied and the following values
in mg/kg were reported in beeswax (0.0001-0.06), honey (0.00001-0.006) and propolis
(0.001-0.07) [326]. Some fungi can grow on cereals, fruits, dried nuts, spices and legumes
produces mycotoxins. The most frequent mycotoxins that found aflatoxins are B1, B2, G1,
G2 and ochratoxin-A. Aflatoxins directly damage DNA and have been shown to cancer
contribution to food contamination, including mycotoxins. Murphy et al., (2006) reported
thet aflatoxins could cause liver damage in the laboratory [327]. Swaileh et al., (2013)
reported that in all analyzed samples occurrence of variable amount of aflatoxins (0.5 to
22µg/kg, mean 12.1µg/kg), with the maximum level in honey from hot and humid semi-
coastal region. There are little information about mycological contamination and
simultaneous co-occurrence of Aspergelous parasiticus or Aspergelous flavus and aflatoxins
detection in honey [328].
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CHAPTER-3
3. MATERIALS AND METHODS
3.1. Collection of Samples
Total hundred (n=100) samples of branded honey, unbranded honey and natural
combs honey were collected. Natural combs honeys (Big bee’s honey, Small bee’s honey,
Beera (Ziziphus), Palosa (Acacia), Sperkay (Trachyspermum), Bekerr (Justicia), Granda
(Carissa opaca ) were directly collected from honey combs, while branded honey (Marhaba,
Qarshi, Versatile, Al-hayat, Young’s, Pak-salman, Langnese) and unbranded honey (Big bee’s
honey, Small bee’s honey, Beera (Ziziphus), Palosa (Acacia), Sperkay (Trachyspermum),
Bekerr (Justicia), Granda (Carissa opaca) were purchased from local market of Khyber
Pakhtunkhwa Pakistan. The samples were brought to PCSIR Laboratories Complex Peshawar.
All the samples in sealed containers were kept in refrigerator till analysis (Figure 3.1, a to
3.5, a).
139
Figure 3.1,a: Honey bee boxes in farm
Figure 3.1,b: Honey bee boxes in farm
Figure 3.1,c: Palosa (Acacia modesta)
140
Figure 3.1,d: Sperkay (Trachyspermum immi)
Figure 3.1,e: Bekerr (Justicia adhatoda)
Figure 3.1,f: Granda (Carissa opaca)
141
Figure 3.1,g: Beera (Ziziphus mauritiana)
Figure 3.2,a: Branded honey samples
142
Figure 3.2, b: Branded honey samples
Beera honey (Ziziphus) Palosa honey (Acacia modesta)
143
Orange honey (citrus) Baker honey (Justicia)
Sp
arky honey (Trachyspermum) Granda honey (Carissa opaca)
144
Small bees honey (Polyfloral) Big bees honey (Polyfloral)
Unbranded honey samples Canes of honey
Figure 3.3,a: Unbranded honey samples
145
Beera honey (Ziziphus) Palosa honey (Acacia modesta)
Bakerhoney (Justicia) Sparky honey (Trachyspermum)
146
Granda honey (Carissa opaca)
Small bee’s honey (Polyfloral) Big bee’s honey (Polyfloral)
147
Figure 3.4,a: Natural comb honey
Figure 3.4,b: Sample collection from comb
Oxytetracycline
148
Streptomycin
Figure 3.5,a: Antibiotics used for honey bees
149
3.2. Chemicals
Analytical and HPLC grade chemicals and reagents were used in the present study. Standards
of streptomycin, oxytetracycline (MP-Biomedicals, LLC Eschwege, Germany), penicillin,
gentamycin (MP-Biomedical. Inc, kirch, France), methanol, acetonitrile, o-phosphoric acid
and ethyl acetate, monopotassium phosphate and citric acid, extra pure were obtained from
Scharlau Spain.Sulfonamide, chloramphenicol, (SC) Semicarbazide, (AMOZ) 3- amino-5 –
morpholinomethyl-2-oxazoliddinone, (AOZ) 3-amino-2-oxazolidinone, (AHD) 1-
aminohydantoin hydrochloride.2-nitrobenzaldehyde, potassium phosphate (Scharlau
Spain), Acetonitrile, ethyl acetate, ammonium hydroxide solutions, acetone, methanol, N,N-
dimethylformamide (DMF) and ammonium acetate were purchased by Merck, Germany.
HPLC solvents were filtered through a 0.45 μM nylon membrane. A BOECO, Germany
balance (BAS 31 Plus), Centrifuge, a Rotary vacuum evaporator (Buchi, Flawil, Switzerland)
were used to prepare samples, extraction and in clean-up procedures. hexane, (Sigma
Aldrich Germany). Methanol, ascorbic acid (Scharlau, Spain), DPPH (1,1-diphenyl-2-picryl
hydroxyl (Sigma Aldrich, Germany) The standards such as chloroganic acid, gallic acid,
vanallic acid, benzoic acid, and syringic acids were obtained from (Sigma Aldrich Germany),
acetic acid, acetonitrile, methanol, n-hexane and hydrochloric acid (Scharlau Spain), fehling’s
solution, fehling A (copper sulphate solution), fehling B (alkaline tartrate solution),
methylene blue indicator, potassium oxalate, lead acetate, sodium hydroxide (Riedel-
Dehaen Germany), chloroform, mueller-hinton agar, alpha nephthole , ferric chloride,
ninhydrin, ammonia, perchloric acid, nitric acid,sodium bisulphate, carrez solution, cupper
carbonate, acetone, xylene, trifluro aceticacid, sulphuric acid (Riedel-Dehaen Germany)
Standards of phytochemicals such as alkaloids, fluorides, tannins,flavonoids, saponins,
thiamine, riboflavin (Sigma Aldrich). Asbestos, ethyl alcohol (Scharlu Spain), Celite (Fluka),
alpha lactos, maltose, beta d- glucose, xylose, fructose, ribose, manose, arabinose, glactose
150
and sucrose were obtained from WINLAB, Wilfred Smith United Kingdom. 1-phenyl-3-
methyl-5-pyrazolone PMP (Sigma Aldrich, Germany), p-amino benzoic ethyl ester,
acetonitrile, potassium di-phosphate, triethylamine, p- tolvidine, barbituric acidwere
purchased from Merck chemicals (Darmstadt, Germany). Aflatoxins standards such as
aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 were procured from bio pure
(Austria).
Prepared standard stock solutions of aflatoxins (1microgram per milliliter) by
diluting in benzene / acetonitrile (98: 2; v/v). Then store in refrigeratorat 4°C, enclosed with
aluminum foil to avoid aflatoxins degradation in ultraviolet light.Acetone / water (85:15; v /
v) , Benzene /Acetonitrile in ratio (98:2; v / v),Chloroform /xylene /acetone in ratio of
60:30:10 (v/v/v), Methanol/ ACN (60:40; v / v), Ethyl acetate/water (80:20; v / v),
Methanol/water (95:5; v / v), Benzene / Acetonitrile (98: 2; v / v).
3.3. Preparation of Reagents
Sodium Hydroxide (0.2 M)
Dissolved 8g sodium hydroxide pellets in distilled water, make up to 1 liter and store
in liter bottom flask.
Ferric Chloride (0.41M)
33g ferric chloride hex hydrate dissolved in distilled water, make up to 300ml and
store in 500ml bottle.
151
Sulphuric Acid (0.03 %)
3ml concentrated sulphuric acid were added to 100ml distilled water and store in
250 ml bottle as 3% sulphuric acid stock solution. Take 20ml of 3% sulphuric acid in a 2 liter
flat bottom flask and make up to 2 liter.
Potassium Hydroxide (0.02M)
2.24g potassium hydroxide was added in distilled water make up to 2liter and store
in flat bottom flask.
Aqueous Acetic Acid (0.1%)
0.1ml of concentrated acetic acid was added to 100ml distilled water.
Ferric Chloride (0.1 %)
0.1ml of ferric chloride was added to 100ml distilled water.
Hydrochloric Acid (1%)
1ml of concentrated hydrochloric acid was added to 99ml distilled water.
Ammonia Solution (1%)
1ml of ammonia was added to 99ml distilled water.
Sodium Hydroxide Solutions (0.1N)
Dissolved 4g sodium hydroxide pellets in distilled water, make up to 1 liter
and store in liter bottom flask.
Hydrochloric Acid Solution (1 N)
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The hydrochloric acid solution was prepared by dissolving 41ml of concentrated
hydrochloric acid in distilled water in 500ml volumetric flask then the solution was diluted
with distilled water up to the mark.
Sodium Hydroxide Solution (0.313 N)
Dissolved 12.52g sodium hydroxide pellets in distilled water, make up to 1 liter and
store in liter bottom flask.
Sodium Hydroxide Solution (1 N)
Dissolved 40g sodium hydroxide pellets in distilled water, make up to 1 liter and
store in liter bottom flask
Sulphuric Acid Solution (0.255 N)
The (0.255 N) sulphuric acid solution was prepared by dissolving 3.48ml of
concentrated sulphuric acid in distilled water in 500ml volumetric flask then the solution was
diluted with distilled water up to the mark.
Sodium Hydroxide (0.02M)
Dissolved 0.8g sodium hydroxide pellets in distilled water, make up to 1 liter and
store in liter bottom flask.
Lead Acetate Solution (45%)
45g of lead acetate were dissolved in distilled water to make volume 100 ml.
Potassium Oxalate Solution (22%)
22g of Potassium oxalate were dissolved in distilled water to make volume 100 ml.
Sodium Hydroxide Solution (20%)
153
20g of sodium hydroxide were dissolved in distilled water to make volume 100 ml.
Potassium Bi-phosphate Solution (0.045%)
0.045g of potassium bi-phosphate was dissolved in distilled water to make volume
100 ml.
Tri-ethylamine Buffer Solution (0.05%)
0.05ml of triethyl amine buffer was dissolved in distilled water to make volume 100
ml.
Sulphuric Acid Solution (50 %)
50ml of sulphuric acid was dissolved in distilled water to make volume 100 ml.
Acetic Acid Solution (0.05 %)
0.05ml of acetic acid was dissolved in distilled water to make volume 100 ml.
Acetic Acid Solution (2 %)
2ml of acetic acid was dissolved in distilled water to make volume 100 ml.
Sodium Bisulphate (0.1 %)
0.1g of sodium bisulphate was dissolved in distilled water to make volume 100 ml.
Buljet’s Reagent
Contain 95ml aqueous picric acid and 5ml 10% aqueous sodium hydroxide
Spand’s Reagent
Contain (4, 5-Dihydroxy-3-(p-sulfophenylazo)-2, 7-naphthalene disulfonic
acid and trisodium salt)
154
Ferric Chloride (0.1 M)
8 g ferric chloride dissolved in distilled water, make up to 300ml and store in
500ml bottle
Hydrochloric Acid (0.1 N)
The hydrochloric acid solution was prepared by dissolving 4.1ml of
concentrated hydrochloric acid in distilled water in 500ml volumetric flask then the
solution was diluted with distilled water up to the mark.
Potassium Dichromate (2%)
2 g of potassium dichromate was dissolved in distilled water to make volume 100 ml.
Potassium Permanganate (5%)
5 g of potassium permanganate was dissolved in distilled water to make volume 100
ml.
Hydrogen Peroxide (30%)
30 ml of hydrogen peroxide was dissolved in distilled water to make volume 100 ml.
Sodium Sulfate (40%)
40 g of sodium sulfate was dissolved in distilled water to make volume 100 ml.
Ethanol (20%)
20 ml of ethanol was dissolved in distilled water to make volume 100 ml.
Sodium Chloride (5%)
5 g of sodium chloride was dissolved in distilled water to make volume 100 ml.
Acetic Acid (20%)
20 ml of acetic acid was dissolved in distilled water to make volume 100 ml.
Methanol (70%)
155
70 ml of methanol was dissolved in distilled water to make volume 100 ml.
Ethanol (50%)
50 ml of ethanol was dissolved in distilled water to make volume 100 ml.
Sodium Hydroxide (10%)
10 g of sodium hydroxide was dissolved in distilled water to make volume 100 ml.
3.4. Determination of Antibiotics
3.4.1. Standards Preparation
Standards of tetracycline, penicillin, streptomycin and gentamycin (0.1g/1mL) were
prepared by dissolving the drug in 60ml methanol and 40ml water. All the solutions were
stored at 4°C and were brought to room temperature before use.
3.4.2. Extraction Procedure for Detection on TLC
The antibiotic residues were extracted by the reported method [329]. Each 5g of
sample was extracted with a mixture of ethyl acetate/water (80:20) by centrifugation at
3000rpm for 10 min and the supernatant was used for spotting on TLC plate for the
detection of antibiotic residue.
3.4.3. Extraction Procedure for HPLC
2 g of honey sample was taken into a 10 ml test tube and intensively shaken with 3
milliliter acetonitrile for 1 minute. The mixture was centrifuged at 5000 rpm for 15 minutes.
The supernatant was collected and dried under nitrogen stream at 40oC. The residue was re-
dissolved in methanol, filter through 0.45um filter membrane and injected 10µl into HPLC
156
system.
3.4.4. TLC Analysis of Antibiotics
The spot of each honey sample and standard of antibiotic were spotted on TLC plate
with the help of automatic TLC spotter [330],[331]. The plates were developed in
methanol/water (95:5), and the detection was carried out by comparing the (Rf) value of
sample with that of standard under UV light of 254nm.
157
3.4.5. HPLC Analysis of Antibiotics
Chromatography instrument model, Hitachi (D-2000 Elite system manager) with a
dual pump (L-2130), Ultraviolet, visible detector (L-2420) and auto sampler (L-2200) was
used for the quantification of antibiotic residue, in which the partition was attained by using
ODS-3 C18 column and column oven (L-2300) (GL Sciences Inc. Tokyo Japan 5µm, 250×4.6
mm). All solvents were filtered through 0.45µm Sartolon Polyimide membrane by a filtration
assembly of (Rocker-300 Model Taiwan) and deduced by ultrasonic cleaner Ceia (Model CP-
104 Italy).
The determination of antibiotic residue in honey samples were carried out according
to a described procedure [332, 333]. Different mixtures of an aqueous mobile phase (A)
acidified water (60%) and organic mobile phase (B) methanol/ acetonitrile (40%) with a flow
rate of 1 ml/min. The compounds were detected at 210-240nm wavelengths. The
quantification was achieved by comparison of the peak area of the sample with that of the
external standard. The identical chromatogram was quantified by the peak area of the
sample with that of standard at a same retention time by giving formula.
Peak area of Sample Conc. of Standard
Sample weight (µg/ml) = x × Standard potency
Peak area of Standard Conc. of Sample
3.5. Standards Preparation for Sulfonamide Antibiotics
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Standards of sulfamethazine, sulfacetamide, sulfathiazole (0.1g/1ml) were prepared
by dissolving the drug in acetonitrile. All the solutions were stored at 4°C and were brought
to room temperature before use.
3.5.1. Extraction Procedure for HPLC
The extraction of sulfonamide were carried out by Andrzej posyniak method [334].
The honey sample (2.5 g) was diluted with 12.5 ml of 0.1 M acetic buffer (pH 5.0) and then
immersed in ultrasonic water bath for 15 min. The whole solution was extracted on an
octadecyl phase chemically bound to the silica gel disposable column under depression of
0.5 bars SPE octadecyl (3ml) columns Deventer, Holland). The SPE-column was
preconditioned with 3 ml of methanol, 3 ml water and finally with 3 ml of acetic buffer (pH
5.0). After percolation of the whole solution, the bed of the column was washed with 3 ml of
acetic buffer (pH 5.0), 3 ml of water and dried under depression for 5 min. The sulfonamides
were eluted with 5 ml of acetonitrile. This extract was dried under a nitrogen stream at 40˚C.
The dry residue was dissolved in 900μl of acetic buffer (pH 3.5); 100μl of 0.2%
fluoroescamine in acetone was added and mixed with a vortex mixer. The sample was ready
to analyze after keeping for 20 min at room temperature.
3.5.2. HPLC Analysis of Sulfonamide Antibiotics
Chromatography instrument model, Hitachi (D-2000 Elite system manager) with a
dual pump (L-2130), fluorescence-detector (L-2485) with excitation wavelength 405 nm and
emission wavelength 495 nm was used to analyze. Auto sampler (L-2200), ODS-3 C18
column (GL Sciences Inc. Tokyo Japan 5µm, 250×4.6 mm) and column oven (L-2300). All
solvents were filtered through 0.45um Sartolon Polyimide membrane by a filtration
assembly of (Rocker-300 Model Taiwan) and deduced by ultrasonic cleaner Ceia (Model CP-
104 Italy).
159
The chromatographic analyses of sulfonamides in honey samples were performed
on ODS-3 C18 column (250x4.6 mm, 5μm) with mobile phase 2% (v/v) acetic acid and
acetonitrile. Flow rate 0.9 ml/min was used for the separation of analytes in gradient mode
at the following program: 0-2 min, acetic acid/acetonitrile (70:30); 2-5 min, acetic
acid/acetonitrile (80:20); 5-12 min, acetic acid/acetonitrile (60:40); 12-19 min, acetic
acid/acetonitrile (70:30). Aliquots of 20 ml were injected into the column. The quantification
was achieved by comparison of the peak area of the sample with that of the external
standard. The identical chromatogram was quantified by the peak area of the sample with
that of the standard at a same retention time by giving formula.
Peak area of Sample Conc. of Standard
Sample weight (µg/ml)= x × Standard potency
Peak area of Standard Conc. of sample
3.6. Chloramphenicol
3.6.1. Standard Preparation
The 100 µg/ml standard solutions were prepared by dissolving of 2.5mg of
chloramphenicol into 25ml acetonitrile in a volumetric flask. Then for working standard
solution (5-100ng/ml) the stock solution was diluted with acetonitrile and water (20:80) in
10ml volumetric flask.
3.6.2. Extraction Procedure for HPLC
Determinations of chloramphenicol in honey the sample extraction were carried out
by Zhao method [335]. 5 gram honey sample was taken into 50ml capped centrifuge tube
added 5ml water and vortex for 3 minutes, added 5ml ethyl acetate to the centrifuge tube,
again vortex for five minutes to mixed properly. Then for five minutes the tube was
160
centrifuged at 3200 rpm. The upper organic layer was carefully transferred through
disposable pipettes to another tube. Supernatant was dry under controlled nitrogen flow at
50ºC temperature, then reconstituted with 5ml water, vortex and sonicated to dissolve
residues completely, the sample was then ready to pass from SPE clean up. 5ml sample
extract passed through the cartage slowly (0.5ml/min), the cottage was rinsed with 5ml
water twice. Discarded the entire effluent, for complete dryness of resins full vacuum was
applied for three minutes the residues were eluted with 5ml of 20:80 methanol / ethyl
acetate at a rate of 26 ml /min. Then collected the effluent in clean tube and dried under
nitrogen streamed at 50ºC the residues were redissolved in 1ml of 20:80 acetonitrile /
water. The sample was vortexed and sonicated to dissolve the residues in the tube
completely.
3.6.3. HPLC Analysis
The presence of chloramphenicol residues in honey samples was carried by HPLC
(Hitachi D-2000 Elite system manager), equipped with dual pumps (L-2130) and auto
sampler (L-2200). The chromatographic separation was achieved using column oven (L-2300)
and Zorbax Eclipse XDB-C8 Column (GL Sciences Inc. Tokyo Japan 3µm, 150×4.6 mm). The
chloramphenicol residues were monitored and determined by using a Diode Array Detector
(L-2455). The filtration assembly (Rocker-300 Taiwan) and ultrasonic cleaner Celia (CP-104
Italy) were used for solvents filtration and degassing.
Analysis of chloramphenicol was achieved by gradient elution of two mobile phases
A and B. Acetonitrile HPLC grade solvent as a mobile phase (A) and water (pH 8.5 adjust with
0.01% ammonia) as a mobile phase (B). The gradient program was set at a flow rate of
0.5ml/min, 0min 20%A-80%B; 0.5ml/min, 0.5min 20%A- 80%B, flow 1.0 ml/min 100%A-
0%B; 8min 100%A-0%B; at 0.5ml/min flow, 10min 80%A-20%B and 12min 80%A-20%B. The
column temperatures were maintained at 30 ºC. The chromatograms were recorded at a
161
wavelength of 280nm (UV-Visible region). The sample injection volumes were 100µL. The
identical chromatogram was quantified by the peak area of the sample with that of the
standard at a same retention time by giving formula.
Peak area of Sample Conc. of Standard
Sample weight (µg/ml) = x × Standard potency
Peak area of Standard Conc. of sample
3.7. Determination of Nitrofuran Antibiotics and Their Metabolites
3.7.1 Preparation of Standard Solutions of Furazolidone and Furaltadone
Furazolidone (FZD), Furaltadone (FTD), as standard were purchased from Sigma-Aldrich. Sock
standard solutions 1000 ppm of furazolidone and furaltadone were prepared by dissolving
each (1000 ppm) one in N,N-dimethylformamide : Acetonitrile (1.5 : 8.5). Working mixed
standard solutions were prepared by half dilution method in acetonitrile.
1000 ppm 100 ppm 50 ppm 25 ppm 12.5 ppm 6.25 ppm
3.125 ppm 1.5625 ppm 0.78125 ppm
3.7.2 Extraction of Honey Sample for Furazolidone and Furaltadone
An amount of 5.0 g honey was weighed into a 250 mL polypropylene copolymer centrifuge
flask. Then, 20 mL of ammonium acetate 79 mM solution (pH 4.6) ammonium hydroxide
solution ≥ 30%. were added to adjust the pH to 8. The mixture was allowed to rest for 15
min. Ethyl acetate (30 mL) was added and stirr for 20 min in a rotary shaker and centrifuged
for 10 min at 3000 rpm. The ethyl acetate layer was collected and evaporated to dryness in a
rotary evaporator at 35 oC and 240 mbar. The resulting extract is reconstituted in 2 mL of a
mixture of acetone and methanol 80:20 (v:v).
162
5 mL of a mixture of acetone:methanol (80:20, v:v) solvent was used to conditioned the Sep-
Pack NH2 cartridge. The reconstituted extract was put onto the cartridge and, then, the
nitrofurans were eluted with 5 mL of the acetone:methanol mixture. The elute was
evaporated to dryness in a vacuum concentrator and the residue was reconstituted with 500
μL of a mixture of ammonium acetate solution 14 mM (pH 4.6):acetonitrile (70:30, v:v).
The resulting solution was filtered through a 0.45 μM PVDF Mini-uniprepTM vial before
chromatography [336].
3.7.3 Chromatographic Conditions
LCMS-MS was performed in Finnigan LCQ Advantage Max, Thermo, coupled to an ion trap
mass analyzer, coupled to a surveyorPlus pump, a surveyorPlus photodiode detector and a
surveyorPlus degasser of Thermo Finnigan, Thermo Electron Corp (San Jose, CA, USA).
Data acquisition was controlled by Xcalibar 2.0 SR2 software. The separation was done by C18
column (5 μM, 15 cm x 4.6 mm) from Merck.
LCMS/MS Analysis:
Column: C18, 5μM (15 cm x 4.6 mm)
Flow rate: 1 mL / min
Mobile phase: Acetonitrile : 20mM Ammonium acetate (30:70)
The mass spectrometer was operated in electrospray positive mode [336].
3.7.4 Material and Methods for AOZ and AMOZ-d5
3-Amino-5-morpholinomethyl-2-oxazolidenone-d5 (AMOZ-d5) was purchase from Sigma-
Merck, 3-amino-2-oxazolidinone (AOZ) and the derivatives of AOZ and AMOZ (2-
nitrophenyl)methylene]-amino]-2-oxazolidinone(2-NPAOZ) and 5-(morpholinomethyl)-3-(2-
163
nitrobenzylidenamino)-2-oxazolidinone(2-NPAMOZ) respectively were prepared in PCSIR
Laboratories Complex, Karachi. Ammonium acetate, anhydrous potassium phosphate dibasic
sodium hydroxide all of ACS reagent grade were purchased from Sigma-Aldrich. Analytical
grade chemicals and solvents were used, expect HPLC grade solvents are used in mobile
phase.
3.7.5 Derivatization of AOZ & AMOZ
2-NPAOZ & 2-NPAMOZ was prepared by taking eqvi. moles of AOZ & AMOZ separately and
2-nitrobenzaldehyde in anhydrous ethanol, and refluxed for 2-3 hr.
ON
NO
O
NO2
H
R
ON
O
H2N
H
R
N OCH2R = (FTD)
R = H (FZD)
N OCH2R =
R = H (AOZ)
(AMOZ)
Derivatization
NO2
CHO
O-Nitrobenzaldehyde
N
NO
O
H
R
NO2
N OCH2R = (2-NPAMOZ)
R = H (2-NPAOZ)
Metabolism
Scheme 1: Derivatization of AOZ and AMOZ-d5
3.7.6 Sample Preparation for Detection of AOZ and AMOZ:
Homogenized honey (1.0g) was weighed into 50 mL polypropylene centrifuge tube. After
weighing 20 mL of water was added and shaked well to dissolve honey. 20 mL of ethyl
164
acetate was added to same centrifuge tube and was centrifuged at room temperature for 15
min. The supernatant was collected in the test tube. Approx. 1 mL of 1000 ppm 2-
nitrobenzaldehyde and 1 mL of diluted HCl were added and vortex for 30 minutes. The EtoAc
was washed twice with 2 mL of water and evaporated to dryness using rotary evaporator at
45oC. To the dried extract 2 mL of methanol was added, and filter through 0.5 μM filters into
autosampler vial [337].
3.7.7. Chromatographic Conditions
LCMS/MS Analysis:
Column: C18, 5μM (15 cm x 4.6 mm)
Flow rate: 1 mL / min
Mobile phase: Acetonitrile : 0.1 % Formic acid (50:50)
The mass spectrometer was operated in electrospray positive mode.
3.8. Determination of Antioxidants
3.8.1. Extraction Procedure
The scavenging of free radical of the extracts were determined against 1,1-
diphenyl-2-picryl hydroxyl (DPPH) as reported [338]. For stock solution 0.01g of each
extract dissolved in 1ml of methanol and further diluted to five different concentrations
(100-600 μg/ml). Same dilutions were also made to the ascorbic acid standard. One ml of
each concentration was mixed thoroughly with a freshly prepared DPPH solution and
incubated for 10 minutes in the dark at room temperature.
3.8.2. UV Analysis
165
After that, each sample was determined by the absorbance of UV at 517 nm
wavelength for its antioxidant activity by the scavenging of free radical of DPPH. The
Scavenging capacity of the sample was compared to that of control (1ml methanol and 2ml
DPPH). The scavenging activity of the free radical of each sample expressed in percent
inhibition using the given equation
Percent (%) DPPH inhibition activity = {(Ab – As) / Ab)} × 100%
Where As; represent the absorbance of test samples, Ab represent the absorbance of the
control reaction or the blank sample [254, 339]. A curve of percent scavenging or percent
inhibition effect against sample concentration was plotted and the concentration where the
scavenging reaches to 50% is its EC-50 value.
3.9. Determination of Phenolic Compounds
3.9.1. Standard Preparation
The standard solution was prepared by dissolving 0.01 g of each phenolic acid such
as chloroganic acid, gallic acid, vanallic acid, benzoic acid and syringic acid in 10 ml
acetonitrile (1mg/ml).Then inject to HPLC.
3.9.2. HPLC Analysis
Determination of phenolic acids were carried out by khan et al method [340].
Each (20 g) sample was mixed with 100 ml of acidic solution pH-2 with Hydrochloric
acid and stirred using magnetically stirred for 15 min. The resultant solutions were
extracted with ethyl acetate. The final solution was then dried under nitrogen steam at
40ºC and stored for further analysis.
High Performance Liquid Chromatography (Hitachi D-2000 Elite system manager) equipped
with two pumps (Models L-2130), auto sampler (L-2200) syringe loading sample injector
valve’s fitted with 20µl sample disk of two hundreds vials and UV-Vis detector (L-
166
2420).Column oven (L-2300) and C-18 column Intersil ODS-3 (Tokyo, Japan 5µm, 250×4.6
mm. GL science Inc). Ultrasonic cleaner Celia (Model CP-104 Italy) and filtering assembly
(Model Rocker-300 Taiwan) were used for degassing and filtration of solvents.
For the determination of phenolic acid the extract was injected into the HPLC
system. The combination of acetonitrile (solvent A), and (0.1%) aqueous acetic acid
solution pH 3.0 (solvent B), (v/v) mobile phase system in the following gradient
elution program for the purpose of chromatographic separation. 0 min, 10%A-90%B;
5min 20%A-80%B; 10min 40%A-60%B; 15min 60%A-40%B and 20min 80%A-
20%B. The chromatograms were obtained at a UV wavelength of 220 nm as all tested
components have major absorption at this wavelength. The flow rate was set at (1.0
ml/minute) and temperature of column was kept at 25°C and the identification of
phenolic acids of each sample, compare their relative retention time with that standard
mixture of chloroganic acid, gallic acid, vanallic acid, benzoic acid and syringic acid.
3.10. Determination of Antifungal, Antibacterial Activity
3.10.1. Test Organisms
The four test organisms such as Candida albicans (ATCC Code 90028), Aspergillus
niger (PCSIR 001), Escherichia coli (35218) and Bacillus cereus (11778)were obtained from
Food Microbiology Laboratory of Pakistan Council of Scientific and Industrial Research
(PCSIR) Peshawar Khyber Pakhtunkhwa, Pakistan (Figure 3.6; a, 3.6; b) [341].
167
Figure 3.6,a: Test organism’s plates
Aspergillus niger Candida albicans
Bacillus cereus Escherichia coli
Salmonella
Bacillus cereus P.aeruginosa
E.coli
A.niger Staphylococcus
C.albicans
168
Figure 3.6,b: Test organism’s images
3.10.2. Preparation of Honey Solutions
Different concentrations of honey solution were prepared in distilled water (10, 30,
50 and 70 % v/v) and were incubated at 37°C for 30 minutes in a shaking water bath.
Incubation was carried at the absence of light because the hydrogen peroxide and glucose
oxidase is photo sensitive [342].
3.10.3. Antimicrobial Activity
Antibacterial activities were determined by disc diffusion method (Mueller-Hinton
Agar) [343]. Antifungal activity of honey samples was evaluated using agar (PDA) disc
diffusion method against test organisms. About 100 µl fresh culture suspension of the test
microorganism was spread on (PDA) Dextrose Agar Plate. The culture concentration was
1×107 CFU/ml. Sterile filter paper 5 mm disc was impregnated with 0.1 mg of honey after
being placed on the surface of the inoculated media agar plates for screening. At 4°C the
plate stood for 2 hours before being incubated optimum condition for 24 hours at 37 °C.
Presence of antimicrobial activity indicates clear inhibition zone around the disc. The
diameter of the inhibition zones were measured in millimeter, including the diameter of
disc. The controls were setup with equivalent quantity of water. First immaculate the honey
samples separately on standard nutrients media with no test organisms so as to investigate
their possible contamination. The diameters of the inhibition zone were determined. All
samples were analyzed in triplicate [344].
3.10.4. Minimum Inhibitory Concentration (MIC)
Determination of minimum inhibitory concentration was done by incorporating
different dilutions of honey (undiluted, 10, 30, 50 and 70 % v/v) into media to check their
efficiency against Candida albicans, Aspergillus niger, Escherichia coli and Bacillus cereus.
Each plate attains final volume of 5 ml including both media and honey, and then incubated
169
for 48 hours at 37 ºC. Minimum inhibitory concentration was determined by finding the
plates with the lowest concentration of honey on which the strain showed no growth [345].
The expression of all (MIC) values is in % (v/v).
Percentage inhibition = 1 – (OD test /OD Control) ×100
For result measurement negative inhibition value were recorded
3.11. Determination of Phytochemicals
The qualitative and quantitative screening test for phytochemicals such as tannins,
phlobatanins, flavonoids, terpenoids, glycoside, saponins, alkaloids, fluorides of branded and
unbranded honey samples were carried out by Official Methods of Analysis [346].
3.11.1 Qualitative Tests
(i) Tannins: 0.5g of honey sample is boiled in 20ml of distal water in a test tube and
filtered, then added 0.1 % ferric chloride and observed green or blue color indicate
the presence of tannin.
(ii) Phlobatanins: 10g of sample is boiled with 1% of hydrochloric acid in test tube red
precipitate produce which indicate the presence of phlobatanins.
(iii) Saponins: 2g of honey sample is boiled with 20 ml distal water in water bath. 10 ml
solution mixed with 5 ml distal water, shacked vigorously and then added 3 drops of
olive oil in the formation of emulsions. Indicate the presence of saponins.
(iv) Flavonoids: 1% of ammonia solution is added to the aqua’s extract of honey samples
in test tubes. Yellow color is produced which shows the presence of flavonoids.
170
(v) Terpenoids: 5g of honey sample is mixed with 2ml of chloroform and concentrated
sulfuric acid carefully added to test tube. It forms a radish layer which indicates the
presence of terpenoids.
(iv) Glycosides: 5g of sample is added to 1ml of sulfuric acid in a test tube and then add
2ml of acetic acid and 1 drop of ferric chloride. It produces a brown ring indicate the
presence of glycosides
(v) Alkaloids: take extract, then added 5 ml hydrochloric acid and picric acid 50ml
produce brown color show the presence of alkaloid.
(vi) Fluorides: take extract and add a drop of ferric chloride it produce green, blue color
which show the presence of fluoride.
3.11.2 Quantitative Procedure
The quantification of phytochemicals such as, tannins, thymine, flavonoids,
terpenoids, glycoside, saponins, alkaloids and fluorides contents in honey was
performed according to the standard protocols.
Tannins
The experiment was performed according to the standard method [347]. One gram
of honey sample was taken in a 100 ml conical flask and 50 ml of double distilled water was
added to it and shaken in a magnetic stirrer for 10 h at room temperature. The solution was
filtered in a 50 ml volumetric flask and made up to the mark using distilled water. 5 ml of
filtered solution was taken in a test tube and 0.0008M potassium hexa ciyano ferrate K4 [Fe
(CN) 6] and 0.1 M FeCl3 in 0.1N HCL was added to it. The absorbance was measured in
171
spectrophotometer at 120 nm wavelength within 10 minutes. A blank was prepared and the
reading was taken in the same wavelength. A standard was prepared using tannic acid to get
100 ppm and measured.
Phlobatanins
Phlobatanins determination was done using the standard method with slight
modification [348]. 50g of honey sample was dispersed in 50 ml ethanolic acid 20%
sodium hydroxide (NaOH) and stirred over a magnetic stirrer for 3 h at room
temperature. The resultant was filtrated through Whatman paper number 1 in a 100 ml
conical flask. 10 ml filtrate was mixed with equal volume of 2% potassium
dichromate solution. As a result, color will develop which was read at 360 nm against
a suitable blank. The phlobatanins content was calculated from the thiamine standard
curve.
Flavonoids
A standard method [349],was followed with slight modification to quantify the total
flavonoids content. 10 g of honey sample were taken in a 250 ml conical flask and 100 ml of
70% methanol were added to it. Magnetic stirrer was used to mix the solution for 3 hours
and filtration of the solution was done using Whatman paper number 1. The remaining
powdered material was re-extracted once again with 70% methanol and filtered in a similar
way. Both the filtrates were mixed and transferred into a crucible and evaporated to dryness
in a hot water bath of 600oC and weighted.
Terpenoids
100g of honey sample were taken separately and soaked in alcohol for 24 hours.
Then filtered, the filtrate was extracted with petroleum ether; the ether extract was treated
as total terpenoids [350].
172
Glycosides
Glycoside content in the honey sample was evaluated using Buljet’s reagent as
described method [351] . 1g of honey sample was soaked in 10ml of 70% alcohol for 2hrs.
Then filtered the sample, The extract obtained was then purified using lead acetate and
sodium bi-phosphate Na2HPO4 solution before the addition of freshly prepared Buljet’s
reagent. The difference between the intensity of colors of the experimental and blank
(distilled water and Buljet’s reagent) samples gives the absorbance and is proportional to the
concentration of the glycosides.
Saponins
Estimation of saponins was done according to slightly modified standard
method [352]. 10 g of honey sample was taken in 250 ml conical flask and 100 ml of
20% ethanol was added to it. The mixture was heated in a hot water bath of 550oC for
5 hours with continuous stirring. The mixture was filtrated through Whatman paper
number 1 and the supernatant liquid was separated. The solid residue was mixed with
20% ethanol and heated in a similar way for about 5 hours. The solution was filtered
and mixed with previously filtered solution. The combined filtered solution was
placed on a hot water bath of 900oC and heated still the volume was reduced to 20%
of its initial volume. The concentrated sample was transferred into a 250 ml
separating funnel and 10 ml of diethyl ether was added to it and shaken vigorously.
The aqueous layer was separated carefully after setting down the solution. The
purification process repeated again. 60 ml of n-butanol extracts were washed twice
with 10 ml of 5% aqueous sodium chloride solution. The remaining solution was
heated in a water bath at 500oC until the solvent evaporates and the solution turns into
semi dried form. The sample was then dried in an oven. This saponins content was
calculated by the following equation:
173
Percentage of saponins = (WEP / WS)*100
Where, WEP = Weight of oven dried end product; WS= Weight of sample taken for test.
Alkaloids
The determination of alkaloids was performed according to the standard method of
Dye et al., (2012) [351]. 5 grams of honey sample was taken into a 250 ml beaker and 250 ml
of 20% acetic acid in ethanol was added to it. Magnetic stirrer was used to mix the solution
for 10 h at room temperature. The solution was filtered using Whatman paper number 1 and
the resultant was placed on a hot water bath (60oC) until the extract volume turns 1/4th of
its initial volume. Concentrated ammonium hydroxide (NH4OH) was added drop wise which
form thick precipitate. Ammonium hydroxide (NH4OH) was added till the formation of the
precipitate was complete. The whole solution was allowed to settle down. The precipitate
was collected by filtration, dried in an oven and weighted.
Fluorides
A standard spand,s method were used to quantify the fluoride contents in honey
samples by spectrophotometer at 580nm wavelength [357]. Standard solution of 10 ml was
taken in 100 ml beaker and added 2 ml of the Spand’s reagent to it, then taken the solution
in the covet and inserted in the sample holder of the spectrophotometer to determine its
absorbance. Absorbance taken of all the standards at 580 nm and standard calibration curve
was plotted thereof. At this moment, the absorbance of the sample was measured in the
same way; the concentration of fluoride was indicated by the calibration curve.
3.11.3. Determination of Chemical Composition
The chemical composition such as ash, pH, moisture, total acidity, electrical
conductivity and total sugars were analyzed by standard methods of AOAC 2000 [353].
174
Instruments
The following instruments and equipment’s were used during the present
work. Top loading balance (Model: GP 3202, Sartorius AG Gottingen), Electrical
Oven (Ontherm Designer Series Oven, Hutt City, New Zealand), Barnstead / Electro
thermal (UK), pH Meter (Model: pH 3110 Set 2, WTW, Germany), Digital
Refractometer (Model: RX-1000, Japan), Electronic Dry Cabinet / Desiccating
Cabinet (Model Dry 60 today’s-instrument) and Atomic Absorption
Spectrophotometer (Model: 2000, Hitachi, Tokyo-Japan)
3.11.3.1. Moisture
2 g of honey sample was taken in a pre-weighed Petri dish and was completely
dry in an oven at 1000C for 4 h. After the sample was completely dried, cooled in
desiccators and weighed again. For determination of moisture content in % using the
following formula.
Moisture (%) = Weight of Sample Taken (g) – Weight of Dried Sample (g) x 100
Weight of Sample Taken (g)
3.11.3.2. Ash
1 g of honey was taken in a pre-weighed crucible and was completely dry in
an oven at 100 0C for 1 h. The sample was charred on low flame and then heated at
600 0C in a muffle furnace until a white ash was obtained with constant weight. The
crucible was then cooled in desiccators and weighed again. The ash content calculated
in percentage using the following formula.
Ash (%) = Weight of the Sample After Ashing (g) ×100
Weight of Sample Taken (g)
3.11.3.3. pH and Total Soluble Solids
175
5 g of honey was added in 150ml distilled water and boiled for 30 min, then
cooled to room temperature and filtered through Linen Cloth filter. The pH of the
filtrate was determined using pre-calibrated digital pH-meter and total soluble solid
contents by digital Refractometer.
3.11.3.4. Total Acidity
10 g of honey was added to in 300ml distilled water and boiled till the volume
was reduced to 250 ml. The mixture was then cooled to room temperature and filtered
through Linen Cloth filter. 20 ml of extract was poured in a titration flask, added to it
a few drops of phenolphthalein indicator and then titrated against 0.1 N sodium
hydroxide solutions. The appearance of light pink coloration showed the end point.
The total acidity (%) of the sample was determined using the following formula.
Total Acidity (%) = Factor × N × Titer × Dilution × 100
SW × SV
SW is the weight of the sample taken (g) and SV is the volume of the sample extract
(20ml) taken for analysis.
3.11.3.5. Crude Fats
2 g of honey was taken in a Thimble and placed in soxhlet extractor. A dried
and pre-weighed round-bottom flask (100ml) was connected to the soxhlet assembly
containing 80ml n-hexane. The assembly was heated with a heating mental for 8 h.
After extraction, the n-hexane was evaporated from the round-bottom flask and the
weight of the round-bottom flask along-with the extract was determined again.The
crude fat contents (%) were calculated using the following formula.
Fat (%) = W1 – W2 × 100
W3
176
Where, W1 is the weight of the round-bottom flask and crude extract (g), W2 is
the weight of empty round-bottom flask (g) and W3 is the weight of samples taken (g)
for analysis.
3.11.3.6. Crude Fiber
2 g of honey sample was taken in a Thimble and deflated using n-hexane as a
solvent in a soxhlet extractor for 8 hours. The defatted sample was boiled for 30 min
in a 200ml sulphuric acid (H2SO4) solution (0.255 N). After boiling, the mixture was
filtered through Linen Cloth filter and washed the residue with distilled water till
obtained the acid free sample. This residue was again boiled using 200mL sodium
hydroxide solution (0.313 N). The mixture was filtered through a dried and pre-
weighed Gooch crucible prepared with asbestos mat. The crucible along-with the
samples was dried in an oven and was weighed and then ignited in a muffle furnace at
600oC for 4 hour and weighed again. For the determination of crude fiber contents
(%) using the following formula.
Crude Fiber (%) = W1 – W2 × 100
W3
Where, W1 is the weight of the dried sample (g), W2 is the weight of the
ignited sample (g) and W3 is the weight of the sample taken (g) for analysis.
3.11.3.7. Reducing Sugar
5 g of honey was mixed with 150ml distilled water. The mixture was boiled
till the volume was reduced to 140ml and then diluted up to 250ml with distilled
water. This mixture was transferred to a beaker (500ml) and a few drops of methylene
blue indicator were added. The sodium hydroxide solution (1 N) was added to its
drop-wise till the appearance of light pink color, then 2ml of lead acetate solution
177
(45%) was added to the mixture and after 10 min 2ml of potassium oxalate solution
(22%) was added to the same mixture. The mixture was then filtered through
Whatman filter paper (No. 5) and marked as Filtrate-A. The Filtrate-A was taken in a
burette. Fehling-A and Fehling-B solutions (each of 5 ml) were taken in two separate
titration flasks and a few drops of methyl blue were added as an indicator and then
titrated against Filtrate-A till the appearance of dark blue color. The reducing sugar
contents (%) were determined using the following formula.
Reducing Sugar (%) = Factor × Dilution × 100
Titer × 1000
Where, Factor is obtained from food analysis manual book of awan et al [354].
Dilution is the total volume of the mixture (250 ml) and Titer is the volume of the
Filtrate-A used during titration (ml).
3.11.3.8. Total Sugar
For the determination of total sugar, 50ml of Filtrate-A was taken in a titration
flask (250ml) and 5 g of citric acid and 50ml distilled water were added into it. The
mixture was boiled for 10 min to invert the sucrose and then cooled to room
temperature. The mixture was then neutralized by drop-wise addition of 20% sodium
hydroxide solution using phenolphthalein as an indicator till the appearance of light
pink color. The light pink color was disappeared by drop-wise addition of
hydrochloric acid solution (1 N). This colorless mixture was taken in a burette and
Fehling-A and Fehling-B solutions (each of 5ml) were taken in two separate titration
flasks and a few drops of methylene blue were added as an indicator and then titrated
against mixture till the appearance of dark blue color. The total sugar contents (%)
were determined using the following formula.
Total Sugar (%) = Factor × Dilution × 100
178
Titer × 1000
Where, Factor is obtained from [354]. Dilution is the total volume of the
mixture (250ml) and Titer is the volume of the Filtrate-A used during titration (ml).
3.11.3.9. Non-Reducing Sugar
The percent content of non-reducing sugar were determined by subtraction of
reducing sugar from the total sugar using the following formula.
Percent content of a Non-reducing Sugars = Total Sugar (%) – Reducing Sugar (%)
3.11.3.10. Minerals
Honey sample of 1 g was taken in digestion tube, and then added per
chloricacid and nitric acid (20 ml). The mixture was heated in the digestion flask upto
250ºC. After complete digestion, 1ml of total mixture remains. This remaining
solution was diluted with 100ml of distal water. The solution concentration was
reported as % (w/v) and in ppm (mg/l) on dry weight and analyzed on atomic
absorption spectrophotometer [55, 355].
3.11.3.11. H.M.F Contents
Five grams of every sample were treated with a clarifying agent (Carrez solution)
transferred to 50ml volumetric flasks and made up to volume with deionized water. The
absorbance of the filtered solution was measured at 284 and 336 nm using a blank produced
with an aliquot of the solution treated with sodium bisulphate (NaHSO4) 0.1 % [57].
3.12. Determination of Carbohydrates
3.12.1. Standard Preparation
179
β-D-lactose, α-D-maltose, β-D-glucose, α-D-xylose, β-D-fructose, β-D-ribose, α-D-
raffinose, α-D-arabinos, α-D-glactose and α-D-sucrose were dissolved in 40ml water. The
mixture was transferred to 100 ml volumetric flask containing 50 ml acetonitrile and make
up to volume with distilled water. Filter through 0.45μm filter membrane, then injected into
HPLC.
3.12.2. Extraction Procedure
Determination of carbohydrates were carried by the reported method of Lv et al.,
(2009) [356]. Each (10g) sample was mixed with 100 ml of deionized water, stirred in a
magnetic stirrer for 15 min. The carbohydrate solution was extracted by 1-phenyl- 3-methyl-
5-pyrazolone (PMP) and p-amino benzoic ethyl ester. 1-Phenyl-3-methyl-5- pyrazolone
(PMP) were used for derivatization. Then the sample extract was concentrated under
vacuum at 40ºC. The extract was re-dissolved in 10ml water for injection in an HPLC system
using a UV detector set at 190 to 380 mm. For the identification of carbohydrate contents of
each sample, compare their relative retention time with that of a standard mixture of
carbohydrates.
3.12.3. HPLC analysis
HPLC (Hitachi D-2000 Elite system manager) equipped with two pumps (Models L-
2130), auto sampler L-2200fitted with a 20µl sample loop of 200 vials and UV-VIS detector L-
2420. The chromatographic separation was achieved using column oven L-2300 and
analytical column Intersil ODS-3 C18 (GL Sciences Inc. Tokyo, Japan 5µm, 250×4.6 mm).
Filtering assembly (Model Rocker-300 Taiwan) and ultrasonic cleaner Celia (Model CP-104
Italy) was used for solvents filtration and degassing.
The combination of acetonitrile (solvent A) and 0.045% KH2PO4 – 0.05% tri
ethylamine buffer (pH7.0) (solvent B) (v/v), mobile phase system in the following gradient
180
elution program for the purpose of chromatographic separation. Gradient elution of 90–89–
86% (B) by a linear decreased from 0–15–40min. The chromatograms were obtained at a UV
wavelength of 190 to 380 nm as all tested components have major absorption at this
wavelength. The flow rate was set at 1.0 ml/min and column temperature was kept at 35 °C
and the identification of each carbohydrate component based on retention times and on
spectral data, quantified by establishing an external standard method.
3.13. Determination of Hydroxy Methyl Furfural
3.13.1. Procedure
The 10 g honey samples were taken and added 20 ml water, then made the volume
50 ml. Take two test tubes add 2 ml honey solution and 5ml p-toluidine to each test tube.
Added 1ml water to one test tube and 1 ml barbituric acid in another test tube, then
measured the absorbance of blank and sample at 550 nm wavelength note the reading on
spectrophotometer [357]. Calculate the H.M.F. on the given formula
H.M.F (mg/100g) = absorbance/cell path length*19.2
Where 19.2=factor
Percent increase in HMF contents was calculated as;
Final concentration – initial concentration ×100
3.13.2. Thermal Treatment of Honey
181
Firstly, 100 g of each honey sample (branded, unbranded and natural comb) was
taken separately in a small glass beaker and kept on the gas stove. The HMF concentration
was determined after heating for different time period (2, 5, 7, 9 and 12 minutes). Similarly
100 g of each honey sample (branded, unbranded and natural comb) was kept separately at
different temperature (10, 20°C) at room temperature and (35, 50 and 70 °C) in electric
oven. The HMF concentration was determined after different time period (2, 5, 7, 9 and 12
minutes) storage.
3.14. Determination of Contaminants
3.14.1. Aflatoxins Extraction Procedure
Total aflatoxins (B1, B2, G1 and G2) was determined by the standard AOAC analytical
method, with thin layer chromatography (TLC) technique AOAC 2000 [358]. Honey sample
of 50gram was blended for three minutes with a 250ml solution of acetone / water (85:15
volume /volume), then filtered by Whatman filter paper. In 400 ml beaker 150ml of filtrate
was collected. Added 170ml sodium hydroxide (0.02M) and 3g basic copper carbonate
powdered along with 30ml ferric chloride solution to the filtrate, mixed properly and
transferred to 600ml beaker. Then filtered the solution mixture and shifted 150ml to
separating funnel of 500ml. The addition of (0.03 %) sulfuric acid and then extracted with
10ml chloroform twice. Lower layer of chloroform was transferred to another separating
funnel. Added potassium hydroxide (0.02M) spins gently for 30 seconds and left it for layer
separation. About 8ml extract of chloroform was collected in a vial. Then dried the vial by
evaporated the chloroform from extract through the gentle flow of nitrogen gas and
temperature of 45oC on heating bath. The dry residues of aflatoxins were obtained.
3.14.2. TLC Analysis
182
Determination of aflatoxins the obtained dried residues were re-dissolved in 200 µl
solution of benzene/acetonitrile in the ratio 98:2 (v/v). Then spotted known concentration
on TLC plate and subjected to 45 minutes to develop. In glass tank plates were developed
with a solution of chloroform/xylene /acetone in a ratio of 60:30:10 (v/v/v). After that
under long wave UV light (wavelength of 365 nm) the spot on plate were observed. By visual
comparison concentration was observed with aflatoxins standard spots. The aflatoxins
identity was confirmed by spraying of 50 % sulphuric acid solution and trifluro acetic acid
reaction [359].
3.14.3. Determination of Heavy Metals
3.14.3.1. Extraction Procedure
One gram of honey samples was taken and transferred to digestion flask then added
about 20ml of perchloric acid and nitric acid. This mixture was heated on 250 °C in the
digestion tube. After digestion 1ml digested solution were diluted up to 100 ml with distilled
water [355].
3.14.3.2. Atomic Absorption Spectrophotometer Analysis
The concentration of heavy metals was analyzed on atomic absorption
spectrophotometer [360]. The toxic and heavy metals estimation such as copper, nickel,
mercury, cadmium, lead and manganese was carried out by atomic absorption
spectrophotometer model (Hitachi zee man Z- 8000 Japan). Equipped with (Hollow cathode
lamp) as radiation source using air acetylene flame for the instrument standardization and
calibration different working standards were used. In each sample the concentration of
different elements was determined. The instrumental conditions were mentioned (Table
3.1).
Table 3.1: Instrumental conditions for the maintenance of each element for FAAS
183
Eleme
nts
Wavelength
(nm)
Slit
width(nm)
Lamp
current(mA)
Air flow
rate (l/min)
Acetylene flow
rate (l/min)
Burner
height(mm)
Cd 240.0 1.3 10 9.5 2.0 10.0
Cu 324.8 1.3 7.5 9.5 2.0 7.5
Pb 334.0 0.2 7.0 9.5 2.0 10.0
Ni 232.0 0.2 10.0 9.5 2.0 10.0
Mn 280.0 1.3 10.0 9.5 2.0 10.0
Co 250.0 0.2 10.0 9.5 2.0 10.0
Hg 255.4 0.2 10.0 9.5 2.0 10.0
184
3.14.3.3. Statistical Analysis
Triplicate determinations were carried out and standard deviation was calculated
from concentration vs. absorbance/ division the calibration curve of the standard elements
was obtained. For each sample data was subjected to one way analysis of variation (ANOVA)
and the main comparison was achieved according to the (post hock test) turkey multiple
comparison test significance value of α=0.01 was used to discriminate significance variation
of the verities with mean [361].
185
CHAPTER-4
4. RESULTS AND DISCUSSION
4.1. Antibiotics
Branded, unbranded and natural honey samples were evaluated for the presence of
streptomycin, oxytetracycline, penicillin and gentamycin antibiotic residues. These antibiotic
residues were detected by thin layer chromatography (TLC) method and the results are
tabulated (Table 4.1) which shows that a total number of 5 samples out of 30 branded and
9 samples out of 39 unbranded were found to be positive, while all 21 samples of natural
honey were found negative. The contamination of oxytetracycline was maximum in
unbranded honey samples, which was about 8.3%, streptomycin and penicillin was 6.66 and
5%, while in branded 5, 6 and 1% of samples were contaminated by penicillin, streptomycin
and oxytetracycline. Whoever gentamycin has not detected in any sample. Positive samples
were proceeds for quantification by HPLC. Retention time of oxytetracycline was 5.63, 2.60
minutes for streptomycin and 10.96 minutes for gentamycin. The quantification of these
positive samples was carried out by comparing the peak area of the sample with that of
targeted standard.
In Table 4.2, branded honey shows that the concentrations (mg/kg) (2.13) of
oxytetracycline were observed in Marhaba honey and (1.54) in Qarshi honey, while not
detected in versatile honey, Al-hayat honey and Langnese honey. The concentrations (3.12)
of penicillin were observed in Qarshi honey and (1.42) in Marhaba honey, while not detected
in versatile honey, Al-hayat honey and Langnese honey. The concentrations (3.12) of
streptomycin were observed in versatile honey, while not detected in Marhaba honey,
Qarshi, Al-hayat honey and Langnese honey samples. In Table 4.3, unbranded honey shows
that the concentrations (6.42) of oxytetracycline were observed in Palosa honey, (3.32) in
186
Berra, (2.34) in Sperkay honey, (2.11) in Small bee’s honey and (1.12) in Big bee’s honey
respectively. The concentrations (4.86) of penicillin were observed in Berra honey, (3.43) in
Small bees honey and (1.76) in Big bee’s honey samples, while not detected in Palosa and
Sperkay honey samples. The concentrations (6.65) of streptomycin were observed in Big
bee’s honey, (1.12) in Berra honey, (2.21) in Palos honey and (2.04) in Small bee’s honey
samples, while not detected in Sperkay honey sample. (Table 4.4) shows that the antibiotics
residues were not detected in any samples of natural comb honey (Figure 4.1,d ; 4.1,e). The
maximum contamination of antibiotics residues were calculated 35.31mg/kg from total
positive unbranded honey samples, while in branded honey samples 9.63mg/kg and natural
honey 0mg/kg were recorded.
The major problem which persists in honey is the occurrence of antibiotic residues which
is present due to broad use of antibiotics for the treatment of different disease. In this study,
I focused on the detection of those major antibiotic residues which are mostly used by the
beekeepers for the treatment of different diseases i.e. tetracycline, penicillin, streptomycin
and neomycin. The detection of these antibiotic residues was carried out by thin layer
chromatography AOAC [206]. The quantification of these compounds were done by different
chromatography technique of HPLC method reported by Bohm et al., (2012) [362].
187
Table 4.1: Detection of antibiotic residues in honey samples
Samples No of samples Penicillin Streptomycin Oxytetracycline Gentamycin Total
Branded 40 2* (5.0%) 2 (2.5%) 2 (5.0%) 0(0%) 6(12.5%)
Unbranded 39 3 (5.0%) 4(6.66%) 5 (8.3%) 0(0%) 12(20%)
Natural 21 0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
Total 100 5(5.0%) 6(6.0%) 7 (7.0%) 0(0%) 18(18%)
* Positive sample
Table 4.2: Concentration of antibiotics residues in branded honey samples (mg/kg)
S. No Compounds Branded honey samples
Marhaba Qarshi Versatile Al-Hayat Langnese
1 Oxytetracycline
2.13 1.54 ND ND ND
2 Penicillin
1.42 3.12 ND ND ND
3 Streptomycin
ND ND 1.42 ND ND
4 Gentamycin
ND ND ND ND ND
5 Total
3.55 4.66 1.42 ND ND
ND: Not Detected
188
189
Table 4.3: Concentration of antibiotics residues in unbranded honey samples (mg/kg)
S. No Compounds Unbranded honey samples
Big bee’s
honey
Small bee’s
honey
Berra
Palosa
Sperkay
1 Oxytetracycline
1.12 2.11 3.32 6.42 2.34
2 Penicillin
1.76 3.43 4.86 ND ND
3 Streptomycin
6.65 2.04 1.12 2.21 ND
4 Gentamycin
ND ND ND ND ND
5 Total
9.53 7.85 9.3 8.63 2.34
ND: Not Detected
Table 4.4: Concentration of antibiotics residues in natural honey samples (mg/kg)
S. No Compounds Natural honey samples
Big bee’s
honey
Small bee’s
honey
Berra
Palosa
Sperkay
1 Oxytetracycline
ND ND ND ND ND
2 Penicillin
ND ND ND ND ND
3 Streptomycin
ND ND ND ND ND
4 Gentamycin
ND ND ND ND ND
190
5
Total ND ND ND ND ND
ND: Not Detected
There are different other peak in each HPLC chromatograms of a honey sample having
tetracycline, Penicillin and streptomycin, which is not concerned with our research so are
not identified (Figure4.1,a; 4.1,b; 4.1,c).
Several international reports are available about antibiotic residues in honey. Fifty
honey samples were screened for antibiotic residue. 3% contaminated by tetracycline, 4% by
Penicillin, 2.85% by Streptomycin, while gentamycin and neomycin were not found in any
analyzed samples [119]. Verdon et al., (2005) reported that the residue of sulfonamides in 3
out of 72 samples, tetracycline in 2 out of 72 and streptomycin was found in 4 out of 284
samples. Residues of chloramphenicol and lactam antibiotics were not found, while in
imported honey samples sulfonamides in 31 out of 98 samples, chloramphenicol 40 out of
85 samples, streptomycine 51 out of 102 samples and tetracycline were detected in 29 out
of 98 samples [363]. 19% of the samples have found to be contaminated by the residue of
tetracycline while the other antibiotic residues were found in trace amount namely
streptomycin, sulfonamides and ciprofloxacin [245].
Figure4.1a: HPLC chromatogram of honey sample: 5.63 oxytetracycline residues,
other peaks at 3.45, 4.63, and 14.92 are not identified
191
Figure 4.1b: HPLC chromatogram of honey sample: 2.60 penicillin residues, other peaks
at 4.98, 6.78, 12.34, 14.45 and 16.23 are not identified
Figure 4.1c: HPLC chromatogram of honey sample: 10.96 streptomycin residues,
other peaks at 4.61, 7.23, 8.02, and 13.49 are not identified
192
Figure 4.1d: Concentration of antibiotic residues in branded honey samples
Figure 4.1,e: Concentration of antibiotic residues in unbranded honey samples
4.1.1 Sulfonamides
Honey bee larvae are susceptible to American foulbrood or European foulbrood, a
disease caused by the organism Bacillus larvae, which can devastate hives. Sulfonamides are
relatively stable chemotherapeutics known to control this disease but they are not
193
permitted to use for this purpose in most countries because of the potential of sulfonamide
residues to contaminate honey [364]. Use of large amounts of sulfonamides in animal
husbandry particularly as veterinary medicine cause to the hazardous effects on people’s
health and environment [131]. These antibiotics can produce allergic hypersensitivity effects
or toxic reactions to human health. For these reasons, the residues of sulfonamides in the
food chains must keep under control. However, so far, maximum residue limits have been
established for sulfonamide compounds in food of animal origin, but not in honey, at level
0.1 mg/kg within the European Union [132].
This study was focused to evaluate the concentration of sulfonamide residues
(sulfamethazine, sulfacetamide and sulfathiazole) (mg/kg) in branded, unbranded and
natural combs honey of Khyber Pakhtunkhwa Pakistan. The results showed (Table 4.5, 4.6
and 4.7) that the concentrations of these residues were not detected in any sample. The
standard chromatogram of sulfonamides represented (Figure 4.2,a).
Figure 4.2,a: HPLC chromatogram of sulfonamides standard, 13.20 sulfacetamide (SCA),
14.10 sulfamethazine (SMT) and 15.05 sulfathiazole (STZ) were identified.
194
Table 4.5: Concentration of sulfonamide antibiotic in branded honey samples
S. No Compounds Concentration of sulfonamide in branded honey (mg/kg)
Marhaba Qarshi Versatile Al-Hayat Langnese
Pak-Salman
1 Sulfamethazine ND ND ND ND ND ND
2 Sulfacetamide ND ND ND ND ND ND
3 Sulfathiazole ND ND ND ND ND ND
ND: Not Detected
Table 4.6: Concentration of sulfonamide antibiotic in unbranded honey samples
S. No Compounds Concentration of sulfonamide in unbranded honey (mg/kg)
Big bee’s
honey
Small bee’s
honey
Berra
Palosa
Sperkay
Bekerr
1 Sulfamethazine ND ND ND ND ND ND
2 Sulfacetamide ND ND ND ND ND ND
3 Sulfathiazole ND ND ND ND ND ND
ND: Not Detected
Table 4.7: Concentration of sulfonamide antibiotic in natural honey samples
S. No Compounds Concentration of sulfonamide in natural honey (mg/kg)
Big bee’s
honey
Small bee’s
honey
Berra
Palosa
Sperkay
Bekerr
1 Sulfamethazine ND ND ND ND ND ND
195
ND: Not Detected
2 Sulfacetamide ND ND ND ND ND ND
3 Sulfathiazole ND ND ND ND ND ND
196
4.1.2 Chloramphenicol
Chloramphenicol (CAP) is a broad-spectrum antibiotic previously used in veterinary
medicine. Recently, some honeys on the international market have been found
contaminated with CAP residues. The EU prohibits the use of CAP as a veterinary drug for
food producing animals. For CAP maximum residue limit (MRL) could not be set. Therefore, a
minimum required performance level (MRPL) was set at 0.3 mg/kg [365].
Brambilla et al., (2012) reported that (CAP) has potentially carcinogenic, which
makes it an unacceptable substance for use with any food producing animals, including
honey bees. Chloramphenicol is anticipated to be a human carcinogen and genotoxic from
studies in humans. It is toxic to blood, kidney, liver. Repeated or prolonged exposure to
chloramphenicol can lead to target organ damage, bone marrow toxicity. The most serious
effect of chloramphenicol is aplastic anemia which is idiosyncratic (rare, unpredictable, and
unrelated to dose) and generally fatal and could presumably be triggered by residues [366].
This study was focused to evaluate the concentration of chloramphenicol residues
(µg/kg) in branded, unbranded and natural combs honey of Khyber Pakhtunkhwa Pakistan.
The results showed (Table 4.8, 4.9 and 4.10) that the concentrations of these residues were
not detected in any samples.
Table 4.8: Concentration of chloramphenicol antibiotic residues in branded honey samples
S. No Compounds Concentration of chloramphenicol in branded honey (µg/kg)
Marhaba
Qarshi Versatile Al-Hayat Langnese Pak-
Salman
1 Chloramphenicol ND
ND ND ND ND ND
ND: Not Detected
197
Table 4.9: Concentration of chloramphenicol antibiotic residues in unbranded honey samples
S. No Compounds Concentration of chloramphenicol in unbranded honey (µg/kg)
Big bee’s
honey
Small
bee’s
honey
Beera
Palosa
Sperkay
Bekerr
1 Chloramphenicol ND ND ND ND ND ND
ND: Not Detected
Table 4.10: Concentration of chloramphenicol antibiotic residues in natural honey samples
S. No Compounds Concentration of chloramphenicol in natural honey (µg/kg)
Big bee’s
honey
Small
bee’s
honey
Beera
Palosa
Sperkay
Bekerr
1 Chloramphenicol ND ND ND ND ND ND
ND: Not Detected
4.1.3 Nitrofurans
Nitrofurans are broad-spectrum antibiotics used to treat bees and other animals with
bacterial infections. As a result of dosing bees with these antibiotics their metabolites are
sometimes found in honey. Apiculture relies on antibiotics to prevent disease propagation
through the densely populated bee colonies. The overuse of antibiotics in honey bee
colonies can cause high levels of residues in honey products, which becomes a public health
issue. Additionally, bacteria that have developed resistance to the applied antibiotics can
pose an increased threat to both human and animal health. Consequently, antibiotics
become less effective against bacteria and there will be fewer alternatives available for the
198
successful treatment of infection. Jenkins et al ., (2005) reported that unscrupulous
producers search for these alternative antibiotics such as nitrofuran to treat disease [367].
The nitrofuran derivatives furazolidone, nitrofurazone, nitrofurantoin, furaltadone are
antibacterial drugs which were used in veterinary practice to treat infections of the urinary
tract, digestive system and skin, and were also used as food preservatives. The antibacterial
action of nitrofurans covers a broad spectrum of micro-organisms (Streptococcus,
Staphylococcus, Gram-negative rods). Nitrofurans also have antiprotozoal and fungicidal
properties. Besides their pharmacological value, nitrofurans elicit numerous side effects like
mutagenicity, carcinogenicity, damage to the lungs and cardiac muscle. In animal organisms
nitrofurans are metabolized quite quickly, so their metabolites are used to monitor
nitrofuran residues as residue biomarkers because of their long half-life in animals (1.9–3.8
weeks) [368]. The respective biomarkers of furazolidone, nitrofurantoin, nitrofurazone and
furaltadone are the metabolites 3-amino-2-oxazolidone, 1-aminohydantoin, semicarbazide,
and 3-amino-5-morpholinomethyl-2-oxazolidone. The administration of nitrofuran to
animals destined to be human food has been prohibited in the EU since 1997, because the
metabolites of this compound can accumulate in animal tissues and their products for a long
time, even after the conclusion of treatment [369].
This study was focused to evaluate the concentration of nitrofurans and their
metabolites residues (furazolidone, furaltadone, nitrofurantoin and nitrofurazone) (ng/kg) in
branded, unbranded and natural combs honey of Khyber Pakhtunkhwa Pakistan. The results
showed (Table 4.11, 4.12 and 4.13) that the concentrations of nitofuran antibiotics and their
metabolites residues were not detected in any samples of honey. The standard
chromatogram of Nitrofuran metabolite represented (Figure 4.2,b to 4.2,m).
199
Figure 4.2,b: LCMS-MS chromatogram of Nitrofuran metabolites standard, 3.90 AOZ=3-
amino-2-oxazolidinone; 4.16 AMOZ = 3-amino-5-morpholino-methyl-1, 3-oxa- zolidinone;
were identified.
200
Figure 4.2,c: LCMS-MS chromatogram of Furazolidone AOZ=3-amino-2-oxazolidinone
201
Figure 4.2,d: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-
methyl-1, 3-oxa- zolidinone
202
Figure 4.2,e: Standerd Colibration; Limit of Detection (LOD) and Limit of Quantification
(LOQ) of Furazolidone
203
Figure 4.2,f: Standerd Colibration; Limit of Detection (LOD) and Limit of Quantification (LOQ)
of Furaltadone
204
Figure 4.2,g: LCMS-MS Chromatogram of honey sample
205
Figure 4.2,h: LCMS-MS chromatogram of Furazolidone AOZ=3-amino-2-oxazolidinone
206
Figure 4.2,i: LCMS-MS chromatogram of Furazolidone AOZ=3-amino-2-oxazolidinone
207
Figure 4.2,j: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-
methyl-1, 3-oxa- zolidinone
208
Figure 4.2,k: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-
methyl-1, 3-oxa- zolidinone
209
Figure 4.2,l: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-
methyl-1, 3-oxa- zolidinone in honey
210
Figure 4.2,m: LCMS-MS chromatogram of Nitrofuran metabolites standard,13.0
Nitrofurantoin AHD= 1-aminohydantoin; 13.8 Nitrofurazone SEM = semicarbazide;
were identified.
Table 4.11: Concentration of nitrofuran antibiotics and their metabolites in branded honey
samples
S.
No
Nitrofuran
Antibiotics
Metabolites Concentration of nitrofuran and their metabolites in branded honey
(ng/kg)
Marhaba Qarshi Versatile Al-Hayat Langnese
Pak-
Salman
1 Furazolidone AOZ
ND ND ND ND ND ND
2 Furaltadone
AMOZ ND ND ND ND ND ND
3 Nitrofurantoin
AHD ND ND ND ND ND ND
4 Nitrofurazone SEM ND ND ND ND ND ND
211
ND: Not Detected
212
Table 4.12: Concentration of nitrofuran antibiotics and their metabolites in unbranded
honey samples
S. No Nitrofuran
Antibiotics
Metabolites Concentration of nitrofuran their metabolites in unbranded honey
(ng/kg)
Big bee’s
honey
Small bee’s
honey
Beera
Palosa
Sperkay
Bekerr
1 Furazolidone
AOZ
ND ND ND ND ND ND
2 Furaltadone
AMOZ ND ND ND ND ND ND
3 Nitrofurantoin
AHD ND ND ND ND ND ND
4 Nitrofurazone
SEM ND ND ND ND ND ND
ND: Not Detected
Table 4.13: Concentration of nitrofuran antibiotics and their metabolites in natural
honey samples
S. No Nitrofuran
Antibiotics
Metabolites Concentration of nitrofuran and their metabolites in natural honey
(ng/kg)
Big bee’s
honey
Small bee’s
honey
Beera
Palosa
Sperkay
Bekerr
1 Furazolidone AOZ
ND ND ND ND ND ND
2 Furaltadone
AMOZ ND ND ND ND ND ND
3 Nitrofurantoin AHD ND ND ND ND ND ND
213
4 Nitrofurazone
SEM ND ND ND ND ND ND
ND: Not Detected
4.2. Antioxidants
The methanol extract of branded, unbranded and natural honey samples was
evaluated for their scavenging activity of 1,1-diphenyl-2-picryl hydroxyl (DPPH) free radical
for different concentrations (100, 200, 300, 500 and 600 µg/ml) of honey samples. The
activity in percent (%) of honey samples extracted in methanol and control (vitamin C
standard) was presented (Table 4.14, 4.15 and 4.16). These scavenging activities were
proportional to the concentration of the extract. As the concentration of these compounds
increased the percent scavenging activity also increased, when scavenging reached to 50 %
was its (EC50) value. This (EC50) value inversely related to percent scavenging. The sample
with lower (EC50)value showed higher antioxidant activity [280]. On 1, 1-diphenyl-2-picryl
hydroxyl assay, the (EC50) values of honey samples were also evaluated and presented (Table
4.17, 4.18 and 4.19). It was observed that with increase in concentration of honey the free
radical scavenging activity increase. The (EC50) value was calculated from linear equation
plotted from the different concentration of extracts against the percent scavenging (Figure
4.3,a; 4.3,b; 4.3,c)
214
0
20
40
60
80
100
100 200 300 400 500 600
DP
PH
Ra
dic
al
Sca
ven
gin
g %
Concentration in ug/g
Sample 1 Sample 2 Sample 3 Sample 4 Vitamin-C
Figure 4.3,a: Antioxidant activity of branded honey samples
Figure 4.3,b: Antioxidant activity of unbranded honey samples
215
Figure 4.3,c: Antioxidant activity of natural comb honey samples
1,1-diphenyl-2-picryl hydroxyl is a free radical compound that has been currently
used to determine the radical-scavenging ability of various compounds (Figure 4.3,d). It
is a stable free radical which dissolves in methanol, has purple color and a characteristic
absorption at 517 nm. As antioxidants donate protons to this radical, the purple color
from the 1,1-diphenyl-2-picryl hydroxyl assay solution becomes light yellow resulting in a
decreases in absorbance . The decrease in absorbance is taken as a measure of the
extent of radical scavenging [370, 371].
N+
O-
O
N+
O-
O
N+
O- O
N NH
216
Figure 4.3,d: Structure of 1,1-diphenyl-2-picryl hydroxyl [372].
In Table 4.14, branded sample of honey has DPPH radical scavenging activity. Al-Hayat
showed maximum antioxidant activity (81.26±1.44) at the concentration 600 µg/ml among
four honey samples, whereas the lowest activity (20.22±1.19) was observed at the
concentration 100 µg/ml in Marhaba. In Table 4.15, unbranded, Small bee’s honey showed
maximum antioxidant activity (84.33±1.23) at the concentration 600 µg/ml among all honey
samples, whereas the lowest activity (24.12±1.17) was observed at the concentration 100
µg/ml in Beera honey. The data shows that by increasing the concentration of samples
decrease the initial absorbance of 1, 1-diphenyl-2-picryl hydroxyl. It was also noted that
different phenolic contents including flavonols, flavones, isoflavonoids, phenolic acids and
catechins were present in honey [373]. In Table 4.16, results showed that the natural comb
honey has an outstanding DPPH radical scavenging activity as compared to branded and
unbranded honey samples. Big bee’s honey showed maximum antioxidant activity
(85.22±1.23) at the concentration 600 µg/ml among four honey samples, whereas the
lowest activity (10.11±1.34) was observed at the concentration 100 µg/ml in Beera honey. In
Table 4.17, in branded honey samples, the maximum EC50 values (462) were obtained for
Marhaba honey. Moderate EC50 values (280) and (344) were obtained for Qarshi and
Versatile honey respectively, while Al-hayat honey showed the lowest value (215).
In Table 4.18, unbranded honey samples, the maximum EC50 values (334) were
obtained for Beera honey. Moderate EC50 values (280) and (323) were obtained for Big bee’s
honey and Palosa honey respectively, while Small bee’s honey showed the lowest value
(260). In Table 4.19, in natural comb honey samples, the maximum EC50 value (99) was
obtained for Beera honey. Moderate EC50 values (98) and (79) were obtained for Big bee’s
honey and Small bee’s honey respectively, while Palosa honey showed the lowest value (21).
217
A lower value of EC50 indicates a higher antioxidant activity. EC50 values of natural comb
honey were lower than branded and unbranded honey samples.
Table 4.14: DPPH radical scavenging activity of branded honey samples
Concentration
(µg/ml)
Marhaba Qarshi Versatile Al-hayat Control
(Vitamin C)
100 20.22±1.19 25.43±1.11 24.32±1.13 26.27±1.29 38.43±1.12
200 28.34±1.14 38.16±1.23 34.14±1.26 41.23±1.36 53.65±2.12
300 35.12±1.16 51.28±1.25 41.33±1.35 54.31±1.13 68.87±1.97
400 43.35±1.23 65.35±1.07 55.12±1.24 64.04±1.22 81.23±2.33
500 51.25±1.34 73.13±1.27 62.19±1.00 76.13±1.43 83.54±2.54
600 57.01±1.22 80.42±1.32 81.24±1.37 81.26±1.44 85.54±2.84
218
Table 4.15: DPPH radical scavenging activity of unbranded honey samples
Concentration
(µg/ml)
Big bee’s
honey
Small bee’s
honey
Beera Palosa Control
(Vitamin C)
100 24.45±1.01 26.32±0.45 24.12±1.17 51.12±1.21 38.43±1.12
200 37.54±1.46 34.17±1.32 38.35±1.25 67.21±1.35 53.65±2.12
300 49.27±2.21 55.46±1.11 48.25±0.26 76.15±1.30 68.87±1.97
400 60.43±1.34 61.11±1.36 58.45±1.43 81.23±1.04 81.23±2.33
500 68.16±1.45 70.26±1.49 62.36±1.33 51.35±1.44 83.54±2.54
600 83.24±2.34 84.33±1.23 81.18±2.00 67.43±1.27 85.54±2.84
Table 4.16: DPPH radical scavenging activity of natural comb honey samples
Concentration
(µg/ml)
Big bee’s
honey
Small bee’s
honey
Beera Palosa Control
(Vitamin C)
100 12.33±1.12 18.22±1.15 10.11±1.34 11.19±1.35 38.43±1.12
200 29.25±1.13 33.13±1.18 28.22±1.14 24.22±1.33 53.65±2.12
300 45.14±1.11 47.12±1.12 43.23±1.16 40.16±1.25 68.87±1.97
400 59.25±1.14 62.24±1.14 58.33±1.35 54.13±1.31 81.23±2.33
500 72.34±1.15 74.24±1.33 71.21±1.17 70.21±1.41 83.54±2.54
600 85.22±1.23 84.23±1.35 83.15±1.22 82.11±1.12 85.54±2.84
Table 4.17: DPPH radical scavenging activity of branded honey (EC50 in µg/g)
Samples
Marhaba Qarshi Versatile Al-hayat Control (Vitamin C)
Branded honey 462 280 344 215 160
219
Table 4.18: DPPH radical scavenging activity of unbranded honey (EC50 in µg/g)
Samples Big bee’s
honey
Small bee’s
honey
Beera Palosa Control (Vitamin C)
Unbranded
honey
280 260 334 323 160
Table 4.19: DPPH radical scavenging activity of natural honey (EC50 in µg/g)
Samples Big bee’s
honey
Small bee’s
honey
Beera Palosa Control (Vitamin C)
Natural comb
honey
98 79 99 21 160
4.3. Phenolic Acids
Honey serves as a natural food having great potency of scavenging free
radicals, which provide protection against many infectious diseases like
atherosclerosis and cancer. The antioxidant activity / phenolic compounds greatly
depend on floral source. These phenolic acid content determined by different
techniques [177, 374]. This study was focused to evaluate the concentration of
phenolic Acids (mg/100g) in branded, unbranded and natural combs honey of Khyber
Pakhtunkhwa. In Table 4.20, branded honey samples shows that the maximum
concentration (0.42±0.02) of chloroganic acid were observed in Marhaba honey,
minimum concentration (0.16±0.03) in Al-hayat honey, whereas moderate
concentration (0.20±0.02) in Versatile honey and Langnese honey (0.32±0.02). It is
investigated that phenolic compound in clover honey was (128±11 mg/1kg) or
220
(1.28±11 mg/100g), for honey expressed as milligrams of gallic acid equivalent,
clover honey shows (1.87 mg/100g) equal to (18.7mg/kg) honey (1.1 p-hydroxy
bezoic acid) [375].
Maximum concentration (0.23±0.03) of gallic acid were observed in Versatile
honey, minimum (0.11±0.02) in Langnese honey, whereas moderate (0.19±0.02) level
in Qarshi honey and (0.16±0.03) in Pak-salman. Maximum concentration (0.33±0.01)
of Vanallic acid was found in Langnese honey, minimum concentration (0.28±0.02)
in Versatile, whereas moderate (0.29±0.02) level in Marhaba honey, while not
detected in Pak-salman, Qarshi and Al-hayat honey. The benzoic acid was found
maximum (1.81±0.02) in Langnese honey, minimum (1.21±0.02) in Qarshi, while
moderate (1.26±0.02) in Pak-salman honey, Al-hayat (1.34±0.01), and Marhaba
honey (1.40±0.03). Standard of phenolic acids chromatogram were represented
(Figure 4.4,a).
Figure 4.4a: HPLC chromatogram of phenolic acids standard, 2.05 gallic acid, 6.45
chloroganicacid, 10.15 syringic acid, 12.05 benzoic acid, and 21.52 vanallic
acid were identified.
221
Syringic acid was only found in Langnese honey (0.21±0.03). Yao et al., (2004)
reported that in different floral honeys, the quercetin is common flavonoids. The
mean quercetin contents in lophostemon, bankasia (heath), Helianthus, Melaleuca
(tea tree) and Guioa, honeys collected from Australia were of (0.33±0.03 mg/100 g
honey) [376].
In Table 4.21, among unbranded honey’s sample the maximum concentration
(0.43±0.02) of chloroganic acid were observed in Beera honey, minimum (0.14±0.02)
in Sperkay honey, while moderate (0.34±0.03) in Big bees honey, Small bee’s honey
(0.26±0.010). Vanallic acid was found maximum (0.24±0.01) in Small bee’s honey,
minimum (0.15±0.01) in Beera honey, whereas moderate (0.23±0.02) in Big bee’s
honey and Palosa honey (0.16±0.01). Maximum concentration (1.77±0.02) of benzoic
acid were observed in Beera honey, minimum (1.02±0.01) in Small bee’s honey,
whereas moderate (1.64±0.03) in Bekerr honey and Sperkay honey (1.16±0.02), but
not detected in Palosa and Big bee’s honey (Figure 4.4,b; 4.4,c).
In Table 4.22, natural combs honey shows that the maximum concentration
(0.90±0.01) of chloroganic acid was observed in Sperkay honey. While minimum
(0.57±0.02) in Small bee’s honey, moderate concentration (0.77±0.03) in Big bee’s’
honey, Beera honey (0.63±0.01) and Palosa honey (0.79±0.02) respectively.
Maximum concentration (0.98±0.03) of gallic acid was observed in Small bee’s
honey. Minimum concentration (0.47±0.03) in Big bee’s honey, whereas moderate
concentration (0.92±0.02) in Palosa honey, Beera honey (0.61±0.02) and Bekerr
honey (0.76±0.02) respectively. Maximum concentration (0.71±0.03) of vanallic acid
was observed in Palosa honey, while minimum concentration (0.22±0.02) in Big
bee’s honey, whereas moderate concentration (0.66±0.02) in Sperkay honey, Small
bee’s honey (0.59±0.02), Beera honey (0.55±0.01) and Bekerr honey (0.52±0.01)
222
respectively. Maximum concentration (1.73±0.02) of benzoic acid was observed in
Small bees’ honey. Minimum concentration (1.02±0.02) in Beera honey, whereas
moderate concentration (1.70±0.01) in Palosa honey, Sperkay honey (1.69±0.02) and
Big bees honey (1.66±0.01). Maximum concentration (0.31±0.02) of syringic acid
was observed in Sperkay honey. Minimum concentration (0.07±0.02) in Bekerr
honey, whereas moderate concentration (0.23±0.01) in Small bee’s honey, Palosa
honey (0.14±0.03), Beera honey (0.09±0.01) and Bekerr honey (0.07±0.02)
respectively (Figure4.4,d).
Escuredo et al., (2012) reported that the mean content of phenolic acids (P-
hydroxybenzoic acid and cinnamic acid) were found in Citrus honey samples
(1.08±0.36) mg/100 g honey [377]. Maximum concentration (0.44±0.03) of syringic
acid were observed in Small bee’s honey, minimum concentration (0.17±0.03) in
Palosa honey, while moderate concentration (0.31±0.04) in Sperkay honey, but not
detected in Big bee’s honey, Beera and Bekerr honey.
Table 4.20: Phenolic acids contents of branded honey samples
S. No Compounds Concentration of phenolic acids (mg/100g)
Marhaba Qarshi Versatile Al-hayat Langnese Pak-salman
1 Chloroganic
Acid
0.42±0.02 ND 0.20±0.02 0.16±0.03 0.32±0.02 ND
2 Gallic
Acid
ND 0.19±0.02 0.23±0.03 ND 0.11±0.02 0.16±0.03
3 Vanallic Acid 0.29±0.02 ND 0.28±0.02 ND 0.33±0.01 ND
4 Benzoic
Acid
1.40±0.03 1.21±0.02 ND 1.34±0.01 1.81±0.02 1.26±0.02
5 Syringic Acid ND ND ND ND 0.21±0.03 ND
223
ND: Not Detected * Mean ± Standard deviation
Table 4.21: Phenolic acids contents of unbranded honey samples
S. No Compounds Concentration of phenolic acids (mg/100g)
Big bee’s Small bee’s Beera Palosa Sperkay Bekerr
1 Chloroganic
Acid
0.34±0.03* 0.26±0.01 0.43±0.02 ND 0.14±0.02 ND
2 Gallic
Acid
0.24±0.02 0.16±0.03 0.11±0.03 0.29±0.02 0.12±0.02 0.22±0.01
3 Vanallic Acid 0.23±0.02 0.24±0.02 0.15±0.01 0.16±0.01 ND ND
4 Benzoic
Acid
ND 1.02±0.01 1.77±0.02 ND 1.16±0.02 1.64±0.03
5 Syringic Acid ND 0.44±0.03 ND 0.17±0.03 0.31±0.04 ND
6 Total
0.81 2.12 2.46 0.62 1.73 1.86
ND: Not Detected * Mean ± Standard deviation
Table 4.22: Phenolic acids contents of natural combs honey samples
S. No Compounds Concentration of phenolic acids (mg/100g)
Big bee’s Small bee’s Beera Palosa Sperkay Bekerr
1 Chloroganic
Acid
0.77±0.03* 0.57±0.02 0.63±0.01 0.79±0.02 0.90±0.01 0.58±0.01
2 Gallic
Acid
0.47±0.03 0.98±0.03 0.61±0.02 0.92±0.02 ND 0.76±0.02
6 Total
2.11 1.40 0.71 1.50 2.78 1.42
224
3 Vanallic Acid 0.22±0.02 0.59±0.02 0.55±0.01 0.71±0.03 0.66±0.02 0.52±0.01
4 Benzoic
Acid
1.66±0.01 1.73±0.02 1.02±0.02 1.70±0.01 1.69±0.02 ND
5 Syringic Acid 0.11±0.02 0.23±0.01 0.09±0.01 0.14±0.03 0.31±0.02 0.07±0.02
6 Total
3.23 4.10 2.90 4.26 3.56 1.93
ND: Not Detected * Mean ± Standard deviation
Figure 4.4,b: Concentrations of phenolic acid in branded honey samples
225
Figure 4.4,c: Concentrations of phenolic acid in unbranded honey samples
Figure 4.4,d: Concentrations of phenolic acid in natural comb honey samples
4.4. Antifungal Antibacterial Activities
The current study represents the microbiological study of Khyber Pakhtunkhwa
honey. According to our study all natural, branded and unbranded honey samples were
tested at different dilutions (10%, 30%, 50% and 70%) showed antifungal activities against
Aspergillus niger, while no activities were observed against Candida albicans (Table 4.23).
Antifungal activity of honey showed that honey stops the growth of Candida albicans [378].
Among branded farm’s honey maximum antifungal activities (10mm) have been shown by
versatile honey at 50 % dilution against Aspergillus niger, while minimum (1mm) by Young’s
honey at 70 % dilution (Figure 4.5,a; 4.5,b). Honey is a natural product having several factors
necessary for antifungal activity [379]. It was also reported by DeMera et al., (2004) that
226
honey from different pathogenic region has different inhibition ability due to the presence
of various concentration of phenolic compound and biomolecules [152].
In Table 4.24, of unbranded honey, maximum antifungal activities (14mm) have been
shown by Palosa and Big bee honey at 50 % dilution against Aspergillus niger, while
minimum (1mm) by Bekerr at 70 % dilution (Figure 4.5,c ; 4.5, d). It has been reported that
the concentration of honey ranged from 30 to 50 % inhabit the growth of many pathogenic
microorganism including Candida albicans [380]. The antifungal growth inhibition of honey is
not related to the osmotic shock resultant from presence of sugar in culture medium [381].
In Table 4.25, of natural comb honey, maximum antifungal activities (15mm and
16mm) have been shown by Small bees honey undiluted and Big bees honey at 50 %
dilution against Aspergillus niger, while minimum (6mm) by Sperkay at 70 % dilution (Figure
4.5, e). It is also stated that the antimicrobial activity of honey raise with increase the
concentration of sugar which lead high osmolarity [283]. In Table 4.26, Candida albicans
show resistance toward branded honey concentration at % (v/v) dilution. Maximum MIC
(88%) at 50 % dilution was found in Marhaba honey and minimum MIC (35%) at 25 %
dilution in Langnese honey (Figure 4.5, f; 4.5, g). Also Aspergillus niger shows the maximum
MIC (90%) in undiluted Marhaba and minimum MIC (3%) in Langnese honey.
In Table 4.27, resistance of Candida albicans has been observed against the
unbranded honey samples. The maximum MIC (93%) at undiluted Big bee’s honey, while
minimum MIC (34%) at 25 % dilution in Granda honey. Aspergillus niger shows maximum
MIC (93%) in Big bees’ honey and minimum MIC (4%) in Granda (Figure 4.5, h). The
conventional treatment is limited for fungal diseases, the reason is due to the limited
variety of the currently antifungal drug, the expensive treatment were needed for
227
prolong therapy. Jessup et al., (2000) reported that the exposure of superficial mycosis to
antifungal drug but the result showed variation [382].
In Table 4.28, Candida albicans show resistance toward natural comb honey concentration
at % (v/v) dilution. The maximum MIC (98%) at undiluted Big bee’s comb honey and
minimum MIC (40%) at 25 % dilution in Granda honey. Also Aspergillus niger shows the
maximum MIC (97%) in undiluted Big bee’s comb honey and minimum MIC (16%) in Granda
comb honey, while at 12% dilution the MIC were ≥100 for all samples (Figure 4.5, I; 4.5, j).
In Table 4.29, branded honey samples, were tested at different dilutions (10%, 30%,
50% and 70 % v/v) showed antibacterial activities against E. coli.Maximum activity (34mm) in
Qarshi and minimum (1mm) were found at 10% dilution in Langnese honey, while the
antibacterial activities against Bacillus cereus showed maximum sensitivity (31mm) at 50 %
dilution in Young’s honey, while minimum sensitivity (1mm) at 50 % dilution in versatile
honey (Figure 4.5, k; 4.5, l). Antibacterial activity of honey is due to the presence of
hydrogen peroxide but some other antibacterial factor as inhabin, like super saturated
solution of sugar which play important role in osmotic property of honey [383, 384].
In Table 4.30, unbranded honey samples were tested at different dilutions (10%,
30%, 50% and 70% v/v) showed antibacterial activities against E. coli.Maximum activity
(35mm) in Big bees honey and minimum activity (2mm) were found at 10% dilution in Palosa
honey. Antibacterial activities against Bacillus cereus showed the maximum activity (34 mm)
at 50% dilution in Big bees honey, while minimum activity (1 mm) at 10% dilution in Granda
honey and the remaining dilutions of honey samples showed moderate sensitivity (Figure
4.5, m; 4.5, n). It has been reported that honey has greater inhibitory effect on gram
negative bacteria E. coli then other test organisms and honey may have high potential as like
therapeutic honey [287]. In Table 4.31, natural comb honey samples were tested at different
dilutions (10%, 30%, 50% and 70% v/v) showed antibacterial activities against E. coli.
228
Maximum activity (44mm) in Big bee’s honey undiluted and minimum activity (7mm) were
found at 10% dilution in Palosa honey. Antibacterial activities against Bacillus cereus showed
the maximum activity (37mm) in Small bee’s honey undiluted, while minimum activity
(6mm) at 10 % dilution in Bekerr honey and the remaining dilutions of honey samples
showed moderate sensitivity (Figure 4.5, o; 4.5, p).
Inhibitory activity of honey dilution depends on different bacteria species. The
concentration of hydrogen peroxide in different honey show varying antimicrobial effect
[385]. Similarly E. coli showed resistance in branded honey concentration at % (v/v) dilution
(Table 4.32). Maximum MIC (92%) in Qarshi honey and minimum MIC (3%) at 12% dilution in
Pak-salman honey. Also Bacillus cereus showed maximum MIC (97%) in Qarshi honey and
minimum MIC (1%) in Pak-salman (Figure 4.5, q; 4.5, r). E. coli and Bacillus cereus were
inhabited at 40 % concentration among six commercial honeys [386].
In Table 4.33, resistance has also been noticed for E. coli against the unbranded
honey concentrations at % (v/v) dilution. Maximum MIC (95%) in Small bee’s honey while
minimum MIC (32%) at 12% dilution in Beera honey. Also Bacillus cereus showed the
maximum MIC (96%) in Small bee’s honey and minimum MIC (9%) in Beera honey (Figure
4.5, s; 4.5, t and 4.5, u). In Table 4.34, resistance has also been noticed for E. coli against the
natural comb honey concentrations at % (v/v) dilutions. Maximum MIC (93%) in Small bee’s
comb honey undiluted, while minimum MIC (30%) at 12% dilution in Granda honey. Also
Bacillus cereus showed the maximum MIC (94%) in Small bee’s honeys undiluted and
minimum MIC (9%) in Granda honey.
The effect of honey on gram negative bacteria was explained by Escuredo et al., (2012)
[377], that due to low pH and the presence of hydrogen peroxide, antioxidants, phenolic
acids, lysozymes and flavonoids make unsuitable for bacterial growth. Chauhan et al., (2010)
reported that the most disposed bacteria included E. coli and Bacillus cereus with MIC of
229
honey in the range of (0.625 - 5.000 mg/ml) and ZDI (Zone Diameter Inhibition) for isolate
range were (6.94 – 35.95mm) respectively [285].
Table 4.23: Antifungal activity of branded honey against C. albicansand Aspergillus niger
Inhibition zone diameter (mm)
Strains Code Concentration Marhaba Qarshi Versatile Al-
hayat
Young’s Pak-
salman
Langnese
C. albicans ATCC
90028
Undiluted ND ND ND ND ND ND ND
10% ND ND ND ND ND ND ND
30% ND ND ND ND ND ND ND
50% ND ND ND ND ND ND ND
70% ND ND ND ND ND ND ND
Aspergillus
niger
PCSIR
001
Undiluted 9 6 7 6 7 5 6
10% 7 6 8 5 4 6 3
30% 8 7 9 6 6 7 4
50% 9 8 10 8 7 9 5
70% 2 2 2 3 1 2 2
* ND: Not Detected
Table 4.24:Antifungal activity of unbranded honey against C. Albicansand Aspergillus Niger
Inhibition zone diameter (mm)
Strains Code Concentration Big
bees
honey
Small
bees
honey
Beera
Palosa
Sperkay
Bekerr
Granda
C. albicans
ATCC
90028
Undiluted ND ND ND ND ND ND ND
10% ND ND ND ND ND ND ND
30% ND ND ND ND ND ND ND
230
50% ND ND ND ND ND ND ND
70% ND ND ND ND ND ND ND
Aspergillus
niger.
PCSIR
001
Undiluted 10 12 9 11 8 6 9
10% 7 8 7 8 6 7 5
30% 9 8 10 6 6 7 4
50% 14 13 11 14 9 8 7
70% 3 2 2 2 3 1 3
* ND: Not Detected
Table 4.25: Antifungal activity of natural comb honey against C. albicansand Aspergillus niger
* ND: Not Detected
Table 4.26: MIC of branded honey against Candida.albicansand Aspergillus niger % (v/v)
Inhibition zone diameter (mm)
Strains Code Concentration Big
bees
honey
Small
bees
honey
Beera
Palosa
Sperkay
Bekerr
Granda
Candida
albicans
ATCC
90028
Undiluted ND ND ND ND ND ND ND
10% ND ND ND ND ND ND ND
30% ND ND ND ND ND ND ND
50% ND ND ND ND ND ND ND
70% ND ND ND ND ND ND ND
Aspergillus
niger
PCSIR
001
Undiluted 12 15 10 13 11 14 10
10% 9 10 9 11 8 10 8
30% 8 9 12 13 10 8 9
50% 16 13 14 14 12 13 14
70% 9 8 7 8 6 7 9
231
Table 4.27: MIC of Unbranded honey against C. albicansand Aspergillus niger.% (v/v)
Samples C. albicans (ATCC 90028) Aspergillus niger (PCSIR 001)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Big bee’s honey 93 89 67 ≥100 93 97 54 ≥100
Small bee’s honey 90 85 60 ≥100 94 95 44 ≥100
Beera 86 82 54 ≥100 90 88 37 ≥100
Palosa 83 77 49 ≥100 85 87 25 ≥100
Sperkay) 78 70 43 ≥100 81 78 14 ≥100
Bekerr 75 67 39 ≥100 73 72 8 ≥100
Granda 73 61 34 ≥100 70 69 4 ≥100
Samples C. albicans (ATCC 90028) Aspergillus niger (PCSIR 001)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Marhaba 88 73 65 ≥100 90 92 45 ≥100
Qarshi 84 75 60 ≥100 88 87 36 ≥100
Versatile 81 77 53 ≥100 83 84 24 ≥100
Al-hayat 76 72 49 ≥100 81 80 14 ≥100
Young’s honey 73 68 43 ≥100 77 79 9 ≥100
Pak-salman 70 64 40 ≥100 72 74 5 ≥100
Langness 69 60 35 ≥100 69 70 3 ≥100
232
Table 4.28: MIC of natural comb honey against C. albicans and Aspergillus niger % (v/v)
Samples C. albicans (ATCC 90028) Aspergillus niger (PCSIR 001)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Big bee’s honey 98 92 71 ≥100 97 96 57 ≥100
Small bee’s honey 94 88 67 ≥100 95 93 48 ≥100
Beera 89 85 58 ≥100 92 86 42 ≥100
Palosa 85 79 53 ≥100 88 82 37 ≥100
Sperkay 83 74 47 ≥100 83 77 31 ≥100
Bekerr 77 70 43 ≥100 76 71 25 ≥100
Granda 75 67 40 ≥100 72 64 16 ≥100
Table 4.29: Antibacterial activity of branded honey against E. coli and Bacillus cereus
Inhibition zone diameter (mm)
Strains Code Concentration Marhaba Qarshi Versatile Al-
hayat
Young’s Pak
salman
Langnese
E. coli ATCC
35218
Undiluted 32 34 31 24 27 23 31
10% 2 2 2 3 2 5 1
30% 9 6 7 5 7 9 7
50% 10 9 11 8 9 10 9
70% 14 10 12 11 13 10 12
Bacillus
cereus
ATCC
11778
undiluted 20 18 21 19 25 17 22
10% 2 5 3 1 6 2 4
30% 8 7 9 12 10 13 9
50% 33 20 24 23 31 19 14
233
70% 2 2 1 4 3 2 2
Table 4.30: Antibacterial activity of unbranded honey against E. coli and
Bacillus cereus
Inhibition zone diameter (mm)
Strains Code concentration Big
bee’s
honey
Small
bee’s
honey
Beera
Palosa
Sperkay
Bekerr
Granda
E. coli
ATCC
35218
Undiluted 35 32 33 23 24 19 17
10% 4 7 5 2 3 5 3
30% 11 14 9 12 13 10 9
50% 15 17 11 16 18 13 12
70% 17 19 14 18 21 17 15
Bacillus
cereus
ATCC
11778
Undiluted 30 27 29 19 20 18 14
10% 4 2 5 2 3 6 1
30% 9 11 8 12 13 7 8
50% 34 31 26 21 24 19 13
70% 2 3 5 4 2 5 2
234
Table 4.31: Antibacterial activity of natural comb honey against E. coli and
Bacillus cereus
Inhibation zone dimeter (mm)
Strains Code Concentration Big
bee’s
honey
Small
bee’s
honey
Beera
Palosa
Sperkay
Bekerr
Granda
E. coli
ATCC
35218
Undiluted 44 39 37 38 36 33 26
10% 9 9 8 7 8 9 8
30% 17 16 9 13 14 12 10
50% 18 19 14 17 19 15 16
70% 16 18 13 15 19 16 14
Bacillus
cereus
ATCC
11778
Undiluted 35 37 33 29 31 27 24
10% 7 8 8 9 8 6 7
30% 12 13 10 14 17 13 15
50% 34 33 29 24 27 22 21
70% 9 9 8 7 8 9 8
Table 4.32: MIC of branded honey against E.coli and Bacillus cereus (v/v %)
Samples E. coli (ATCC 35218) Bacillus cereus (ATCC 11778)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Qarshi 92 87 55 25 97 88 56 41
Marhaba 89 80 49 23 94 83 53 36
Versatile 85 76 45 21 92 80 49 31
Al-hayat 79 72 38 18 89 77 43 29
235
Young’s honey 76 67 35 12 85 74 41 24
Langnes,s 73 63 32 7 81 71 39 20
Pak-salman 70 60 29 3 78 65 34 15
Table 4.33: MIC of Unbranded honey against E. coli and Bacillus cereus (v/v %)
Samples E. coli (ATCC 35218) Bacillus cereus(ATCC 11778)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Small bee’s honey 95 91 70 57 96 81 57 34
Big bee’s honey 92 88 67 53 90 73 52 31
Beera 90 86 58 50 87 69 49 26
Palosa 87 77 54 48 74 62 42 22
Sperkay 82 74 49 41 68 57 39 19
Bekerr 78 71 46 38 63 51 34 13
Granda 75 65 40 32 60 48 30 9
236
Table 4.34:MIC of natural comb honey against E. coli and Bacillus cereus
Samples E. coli (ATCC 35218) Bacillus cereus (ATCC 11778)
Concentration of samples Concentration of samples
Undiluted 50% 25% 12% Undiluted 50% 25% 12%
Small bee’s honey 93 90 60 58 94 82 58 30
Big bee’s honey 91 87 59 56 88 76 54 28
Beera 89 83 57 51 84 70 49 25
Palosa 84 76 50 47 77 63 40 20
Sperkay 80 70 47 42 68 55 37 17
Bekerr 73 67 44 36 64 50 31 12
Granda 70 62 41 30 61 44 29 9
Figure 4.5,a: Antifungal activity of branded honey samples against Aspergillusniger
237
Figure 4.5,b: Antifungal activity of unbranded honey samples against Aspergillus niger
Figure 4.5,c: Antifungal activity of natural comb honey samples against Aspergillus niger
238
Figure 4.5,d: Minimum inhibitory concentration of branded honey samples against
C.albicans
Figure 4.5,e: Minimum inhibitory concentration of branded honey samples against
Aspergillus niger
239
Figure 4.5,f: Minimum inhibitory concentration of unbranded honey samples against
C.albicans
Figure 4.5,g: Minimum inhibitory concentration of unbranded honey samples against
Aspergillus niger
240
Figure4.5,h: Minimum inhibitory concentration of natural comb honey samples against
C.albicans
Figure4.5,i: Minimum inhibitory concentration of natural comb honey samples against
Aspergillus niger
241
Figure4.5,j: Antibacterial activity of branded honey samples against E.coli
Figure4.5,k: Antibacterial activity of branded honey samples against Bacillus cereus
242
Figure4.5,l: Antibacterial activity of unbranded honey samples against E.coli
Figure4.5,m: Antibacterial activity of unbranded honey samples against Bacillus cereus
243
Figure 4.5,n: Antibacterial activity of natural comb honey samples against E.coli
Figure4.5,o: Antibacterial activity of natural comb honey samples against Bacillus cereus
244
Figure4.5,p: Minimum inhibitory concentration of branded honey samples against E.coli
Figure4.5,q: Minimum inhibitory concentration of branded honey samples against Bacillus
cereus
245
Figure4.5,r: Minimum inhibitory concentration of unbranded honey samples against E.coli
Figure4.5,s: Minimum inhibitory concentration of unbranded honey samples against Bacillus
cereus
246
Fig4.5,t: Minimum inhibitory concentration of natural comb honey samples
against E.coli
Figure4.5,u: Minimum inhibitory concentration of natural comb honey samples against
Bacillus cereus
247
248
4.5. Phytochemicals
The study was under taken to evaluate the phytochemicals analysis of branded,
unbranded and natural comb honey samples. The qualitative study of phytochemicals
showed that the branded and unbranded and natural comb honey were composed of
tannins, phlobatanins, flavonoids, terpenoids, glycosides, saponins, alkaloids and fluorides
represented (Table 4.35, 4.36 and 4.37). The concentration of saponins, flavonoids and
tannins confirms the astringent property of honey. This compound can also be effective in
protecting the kidneys [387].
The quantitative analysis of phytochemicals is presented in Table 4.38. It showed
that in branded honey samples the maximum concentration of tannin (0.35±0.07) was found
in Versatile honey sample, while minimum concentration (0.25±0.06) in Young’s honey.
Tannins have also shown potential antibacterial and antiviral effects [388]. Maximum
concentration of Phlobatanins (0.61±0.05) was found in Qarshi, while minimum
concentration (0.39±0.08) in Young’s honey. Maximum concentration of flavonoids
(0.33±0.01) was found in Pak-salman, while minimum concentration (0.18±0.04) in Qarshi
(Figure 4.6, a). Evans et al., (2009) reported that flavonoids have antibacterial, anti-
inflammatory, anti-allergic, antimultagenic, antiviral, antineoplatic, anti-thrombotic and
vasodilatory activities. However, the low amount of alkaloid presence is also indicative of its
harmless effect based on its content [389].
Maximum concentration (0.41±0.06) of terpenoids was found in Langnese honey,
while minimum concentration (0.24±0.03) in Marhaba. Maximum concentration (0.32±0.06)
of glycoside was found in Langnese, while minimum concentration (0.14±0.04) in Al-hayat.
The saponins contents is important source of detergents, surface active agents used in
industrial applications and also possesses beneficial health effects [390]. Maximum
249
concentration (2.44±0.03) of saponins was found in Pak-salman, while minimum
concentration (1.43±0.01) in Versatile. Maximum concentration (0.46±0.06) of alkaloid was
found in Young’s honey, while minimum concentration (0.25±0.03) in Qarshi honey. Evans et
al., (2009) reported that honey containing alkaloids do not feature strongly in herbal
medicine because they are extremely toxic, so low as to be harmless [389]. Maximum
concentration of fluoride (0.34±0.04) was found in Al-hayat, while minimum concentration
(0.10±0.02) in Pak-Salman honey.
In unbranded honey samples (Table 4.39), showed that the maximum concentration
of tannin (0.54±0.04) was found in Beera, while minimum concentration (0.34±0.03) in
Bekerr honey. Maximum concentration (0.76±0.05) of phlobatanins was found in Sperkay
while minimum concentration (0.65±0.09) in Beera. Maximum concentration (0.36±0.08) of
flavonoids was found in Palosa, while minimum concentration (0.27±0.05) in Beera
(Figure4.6, b).
Table 4.35: Qualitative test for phytochemicals in branded honey samples
Parameter Marhaba Qarshi Versatile Al-hayat Young’s Pak-salman Langnese
Tannins _ _ + _ + _ _
Phlobatanins _ + _ _ + _ +
Flavonoids _ + + _ _ + _
Terpenoids + _ _ _ _ + +
Glycosides + _ _ + + _ +
Saponins + _ + _ + + _
Alkaloids _ + _ _ + _ +
Fluorides + _ + + _ + _
+ = Present - = Absent
250
Table 4.36: Qualitative test for phytochemicals in unbranded honey samples
Parameter Big bee’s
honey
Small
bee’s
honey
Beera Palosa Sperkay Bekerr Granda
Tannins + + + + + + +
Phlobatanins _ + + _ + _ +
Flavonoids + + + + + + +
Terpenoids + + + + _ + +
Glycosides + + + + + + _
Saponins + + + + _ _ +
Alkaloids + _ _ _ + + +
Fluorides + + + + _ _ +
+ = Present - = Absent
Table 4.37: Qualitative test for phytochemicals in natural comb honey samples
Parameter Big bee’s
honey
Small
bee’s
honey
Beera
honey
Palosa
honey
Sperkay
honey
Bekerr
honey
Granda
honey
Tannins + + + + + + +
Phlobatanins + + + + + + +
Flavonoids + + + + + + +
Terpenoids + + + + _ + +
Glycosides + + + + + + +
Saponins + + + + + + +
Alkaloids + + + + + + +
251
Flourides + + + + + + +
+ = Present - = absent
Maximum concentration of terpenoids (0.45±0.06) was found in Granda, while minimum
concentration (0.33±0.05) in Big bee’s honey. Maximum concentration (0.47±0.06) of
glycoside was found in Sperkay honey while minimum concentration (0.31±0.08) in Big bee’s
honey. Maximum concentration (3.49±0.07) of saponins was found in Palosa, while
minimum concentration (2.11±0.04) in Granda. Maximum concentration of alkaloid
(0.34±0.02) was found in Big bee’s honey, while minimum concentration (0.22±0.06) in
Bekerr honey. Maximum concentration (0.25±0.04) of fluorides was found in Beera, while
minimum concentrations (0.11±0.03) in Big bees honey.
In natural comb honey samples (Table 4.40) showed that the maximum
concentration of tannin (0.57±0.01) was found in Big bee’s honey, while minimum
concentration (0.37±0.02) in Granda honey. Maximum concentration of phlobatanins
(0.79±0.02) was found in Small bee’s honey while minimum concentration (0.25±0.03) in Big
bee’s honey. Maximum concentration (0.44±0.02) of flavonoids was found in Bekerr honey,
while minimum concentration (0.31±0.01) in Palosa honey. Maximum concentration of
terpenoids (0.48±0.03) was found in Beera honey, while minimum concentration (0.31±0.02)
in Sperkay honey. Maximum concentration (0.51±0.05) of glycoside was found in Small bee’s
honey while minimum concentration (0.25±0.03) in Granda honey. Maximum concentration
(3.83±0.02) of saponins was found in Big bee’s honey, while minimum concentration
(1.55±0.02) in Bekerr honey. Maximum concentration of alkaloid (0.33±0.02) was found in
Small bee’s honey, while minimum concentration (0.13±0.04) in Palosa honey. Maximum
concentration (0.32±0.03) of fluorides was found in Palosa honey, while minimum
concentrations (0.09±0.04) in Beera honey (Figure 4.6, c).
252
253
Table 4.38: Quantitative analysis for phytochemicals in branded honey samples
Parameter
(mg/g)
Marhaba Qarshi Versatile Al-hayat Young’s Pak-salman Langnese
Tannins ----- ----- 0.35±0.07 ----- 0.25±0.06 ----- -----
Phlobatanins ----- 0.61±0.05 ----- 0.59±0.07 0.39±0.08 ----- 0.61±0.01
Flavonoids ----- 0.18±0.04 0.21±0.06 ----- ----- 0.33±0.01 -----
Terpenoids ----- ----- ----- ----- ----- 0.39±0.08 0.41±0.06
Glycosides 0.21±0.05 ----- ----- 0.14±0.04 0.29±0.02 0.32±0.06
Saponins 2.13±0.02 ----- 1.43±0.01 ----- 2.35±0.09 2.44±0.03 -----
Alkaloids ----- 0.25±0.03 ----- 0.27±0.01 0.46±0.06 ----- 0.27±0.02
Flourides 0.14±0.09 ----- 0.23±0.07 0.34±0.04 ----- 0.10±0.02 -----
Total 2.48 1.00 2.22 1.34 3.74 3.43 1.61
* Mean ± S.D
Table 4.39: Quantitative test for phytochemicals in unbranded honey samples
Parameters
(mg/g)
Big
bee’s
honey
Small
bee’s
honey
Beera Palosa Sperkay Bekerr Granda
Tannins 0.43±0.03 0.51±0.05 0.54±0.04 0.41±0.06 0.49±0.07 0.34±0.03 0.39±0.09
Phlobatanins -------- 0.72±0.06 0.65±0.09 -------- 0.76±0.05 -------- 0.66±0.06
Flavonoids 0.28±0.09 0.30±0.06 0.27±0.05 0.36±0.08 0.29±0.04 0.33±0.07 0.37±0.03
Terpenoids 0.33±0.05 0.38±0.04 0.41±0.07 0.39±0.09 -------- 0.42±0.08 0.45±0.06
Glycosides 0.31±0.08 0.33±0.08 0.44±0.09 0.39±0.05 0.47±0.06 0.37±0.07 --------
Saponins 3.24±0.06 3.22±0.07 2.72±0.03 3.49±0.07 -------- -------- 2.11±0.04
254
Alkaloids 0.14±0.02 -------- -------- -------- 0.18±0.07 0.12±0.06 0.17±0.08
Flourides 0.11±0.03 0.23±0.06 0.25±0.04 0.21±0.03 -------- -------- 0.14±0.06
Total 4.84 5.69 5.28 5.29 2.19 1.58 4.29
* Mean ± S.D.
255
Table 4.40: Quantitative test for phytochemicals in natural comb honey samples
Parameters
(mg/g)
Big bee’s
honey
Small bee’s
honey
Beera
honey
Palosa
honey
Sperkay
honey
Bekerr
honey
Granda
honey
Tannins 0.57±0.01 0.55±0.04 0.45±0.02 0.43±0.02 0.52±0.03 0.41±0.03 0.37±0.02
Phlobatanins 0.25±0.03 0.79±0.02 0.69±0.02 0.52±0.03 0.65±0.02 0.56±0.04 0.53±0.02
Flavonoids 0.43±0.02 0.34±0.01 0.32±0.01 0.31±0.01 0.35±0.04 0.44±0.02 0.42±0.01
Terpenoids 0.41±0.06 0.34±0.02 0.48±0.03 0.44±0.03 0.31±0.02 0.46±0.03 0.44±0.01
Glycosides 0.33±0.03 0.51±0.05 0.39±0.01 0.33±0.02 0.41±0.33 0.38±0.01 0.25±0.03
Saponins 3.83±0.02 2.22±0.03 3.25±0.03 3.55±0.01 2.24±0.01 1.55±0.02 3.12±0.04
Alkaloids 0.21±0.04 0.33±0.02 0.22±0.02 0.13±0.04 0.21±0.03 0.14±0.01 0.24±0.02
Flourides 0.13± 0.02 0.21±0.01 0.09±0.04 0.32±0.03 0.22±0.02 0.12±0.03 0.16±0.01
Total 6.16 5.29 5.89 6.03 4.91 4.06 5.35
* Mean ± S.D.
256
Figure4.6,a: Concentration of phytochemicals in branded honey samples
Figure4.6,b: Concentration of phytochemicals in unbranded honey samples
Figure 4.6,c: Concentration of phytochemicals in natural comb honey samples
257
4.6 Chemical Composition
Chemical composition such as moisture, ash, acidity, fiber, fats, sugar, pH, electrical
conductivity, HMF, sucrose content and vitamin C for branded honey samples (Table 4.41)
were reported. Result showed slight variation among the concentration of different
parameters. On the basis of overall chemical composition in branded honey samples the
maximum concentration of crude fiber (0.9±0.05) was found in Young’s honey while
minimum (0.3±0.03) in Marhaba. Maximum concentration of vitamin C (1.77±0.07) was
found in Al-hayat, while minimum (0.10±0.01) in Young’s honey. Maximum concentration of
moisture (29.1±2.0) was found in Young’s honey, while minimum (21.4±4.0) in Qarshi.
Maximum concentration of fats (0.9±0.06) was found in Versatile, while minimum
(0.1±0.02) in Pak-salman. Maximum concentration of pH (4.1±0.2) was found in Marhaba,
while minimum (3.1±0.5) in Young’s honey (Figure 4.6, g). Turhan et al., (2007) reported that
pH values in the range of 3.34 to 4.70, which are normally accepted [391]. Maximum
concentration of acidity (43.7±2.0) was found in Pak-salman while minimum concentration
(36.5±4.0) in Qarshi (Figure 4.6, j). Free acidity shows a mean value of (27.2 meq kg -1 one
reported in Spain [392]. Maximum concentration of ash (0.61±0.07) was found in Pak-
salman while minimum concentration (0.36±4.0) in Qarshi. Mean concentration of ash
reported in samples from Turkey is (0.25 to 0.45%) [391]. Maximum concentration of
electrical conductivity (6.33±0.07) was found in Young’s honey while minimum (1.24±0.02)
in Pak-salman (Figure 4.6, m). Maximum concentration of HMF (13.3±0.2) was found in
Marhaba while minimum (3.4±0.1) in Al-hayat (Figure 4.6, p). Maximum concentration of
reducing sugar (60.6±1.8) was found in Versatile while minimum (39.2±3.7) in Qarshi honey.
Maximum concentration of sucrose (3.9±0.6) was found in Pak-salman while minimum
(1.6±0.8) in Versatile (Figure 4.6, d i; 4.6,d ii).
258
* Mean ± S.D.
Table 4.41: Chemical composition of branded honey samples
Parameters Marhaba Qarshi Versatile Al-hayat Young’s Pak-salmon
Crude fiber
(%)
0.3±0.03* 0.5±0.01 0.8±0.02 0.7±0.09 0.9±0.05 0.7±0.06
Vitamin C
(%)
0.17±0.03 0.32±0.08 0.56±0.04 1.77±0.07 0.10±0.01 0.44±0.03
Moisture
(%)
26.0±2.0 21.4±4.0 27.6±3.0 24.5±2.0 29.1±2.0 24.6±3.0
Fats
(%)
0.7±0.03 0.4±0.04 0.9±0.06 0.3±0.01 0.6±0.07 0.1±0.02
pH
4.1±0.2 3.9±0.1 3.8±0.3 4.0±0.1 3.1±0.5 3.7±0.4
Acidity
(meq/kg)
42.2±2.0 36.5±4.0 41.3±3.0 40.1±6.0 38.4±1.0 43.7±2.0
Ash
(%)
0.23±0.01 0.35±0.08 0.61±0.07 0.16±0.04 0.36±0.09 0.14±0.03
Electr-condc
(mS/cm)
3.43±0.04 3.26±0.03 2.66±0.01 4.36±0.06 6.33±0.07 1.24±0.02
HMF
(mg/kg)
13.3±0.2 13.0±0.5 10.8±0.7 3.4±0.1 5.2±0.6 8.1±0.4
Reducing
sugar (%)
55.8±2.4 39.2±3.7 60.6±1.8 57.9±3.9 44.5±2.7 56.4±4.1
Sucrose
(%)
3.2±0.5 3.1±0.1 1.6±0.8 3.7±0.3 2.6±0.4 3.9±0.6
Total
86.4 65.27 92.67 89.03 77.96 86.28
259
In Table 4.42, unbranded honey sample, maximum concentration of crude fiber
(0.9±0.03) was found in Small bee’s honey while minimum (0.1±0.06) in Big bee’s honey.
Maximum concentration (1.36±0.04) of vitamin C was found in Sperkay while minimum
(0.14±0.02) in Big bee’s honey. Maximum concentration (29.3±2.0) of moisture was found in
Beera, while minimum (22.2±2.0) in Palosa. Maximum concentration (0.8±0.04) of fats was
found in Palosa, while minimum (0.2±0.01) in Small bee’s honey. Maximum value (4.7±0.1)
of pH was found in Beera, while minimum (2.4±0.2) in Sperkay (Figure 4.6, h). Maximum
concentration (41.4±3.0) of acidity was found in Big bee’s honey while minimum (31.4±2.0)
in Bekerr (Figure 4.6, k).
Table 4.42: Chemical composition of unbranded honey samples
Honey
samples
Big bee’s
honey
Small bee’s
honey
Beera Palosa Sperkay Bekerr
Crude fiber
(%)
0.1±0.06 0.9±0.03 0.2±0.08 0.5±0.01 0.3±0.05 0.2±0.04
Vitamin C
(%)
0.14±0.02 1.28±0.07 0.16±0.01 0.41±0.03 1.36±0.04 0.29±0.08
Moisture
(%)
24.1±3.0 26.4±3.0 29.3±2.0 22.2±2.0 23.9±1.0 27.3±3.0
Fats
(%)
0.6±0.09 0.2±0.01 0.4±0.06 0.8±0.04 0.6±0.07 0.3±0.05
pH 3.2±0.4 2.9±0.8 4.7±0.1 3.5±0.9 2.4±0.2 2.9±0.3
Acidity
(meq/kg)
41.4±3.0 32.2±4.0 39.1±2.0 36.2±3.0 37.6±1.0 31.4±2.0
Ash
(%)
0.12±0.05 0.25±0.01 0.53±0.07 0.22±0.02 0.17±0.09 0.32±0.03
Electrical
conductivity
(mS/cm)
2.33±0.01 2.71±0.03 3.11±0.07 3.53±0.02 2.56±0.06 3.28±0.09
HMF
(mg/kg)
14.5±0.4 12.2±0.7 16.4±0.1 15.8±0.6 11.6±0.5 13.1±0.2
260
Reducing
sugar (%)
60.1±3.0 42.5±4.0 76.2±6.0 81.7±9.0 62.3±3.0 59.2±4.0
Sucrose
(%)
1.4±0.7 2.5±0.1 3.6±0.4 1.8±0.8 2.0±0.7 1.7±0.1
Total
86.56 74.03 98.04 99.04 88.53 89.31
* Mean ± S.D.
Maximum concentration (0.53±0.07) of ash was found in Beera, while minimum
(0.12±0.05) in Big bee’s honey (Figure 4.6, e i; 4.6, e ii). Maximum concentration (3.53±0.02)
of electrical conductivity was found in Palosa, while minimum (2.33±0.01) in Big bee’s
honey. HMF represents the freshness of honey and depends on adequate bee hives and
harvest practice. The value was reported in Turkey (4.52+40 mg kg-1) by Terrab et al.,
(2004)[392].
In our samples, maximum concentration of HMF (16.4±0.1) was found in Beera,
while minimum (11.6±0.5) in Sperkay (Figure 4.6, q). Maximum concentration (81.7±9.0) of
reducing sugar was found in Palosa, while minimum (42.5±4.0) in Small bee’s honey.
Reducing sugar has reported in samples from Turkey (71.32 %) [393]. Maximum
concentration of sucrose (3.6±0.4) was found in Beera, while minimum (1.4±0.7) in Big bee’s
honey.
In Table 4.43, natural comb honey samples, maximum concentration (0.8±0.01) of
crude fiber was found in Big bee’s honey while minimum (0.1±0.02) in Sperkay honey.
Maximum concentration (1.55±0.03) of vitamin C was found in Bekerr honey while minimum
(0.21±0.04) in Beera honey. Maximum concentration (21.5±3.0) of moisture was found in
Bekerr honey, while minimum (18.19±2.0) in Small bee’s honey. Maximum concentration
(0.7±0.01) of fats was found in Beera honey, while minimum (0.3±0.02) in Big bee’s honey.
Maximum value of pH (4.8±0.3) was found in Big bee’s honey, while minimum (3.4±0.1) in
261
Small bee’s honey (Figure 4.6, i). Maximum concentration (43.3±1.0) of acidity was found in
Bekerr honey while minimum (32.2±2.0) in Big bee’s honey. Maximum concentration
(0.46±0.02) of ash was found in Sperkay honey, while minimum (0.14±0.03) in Beera honey
(Figure 4.6, f i; 4.6, f ii).
262
Table 4.43: Chemical composition of natural comb honey samples
Parameters Big bee,s
honey
Small bee,s
honey
Beera
honey
Palosa
honey
Sperkay
honey
Bekerr honey
Crude fiber
(%)
0.8±0.01 0.2 ±0.02 0.3±0.01 0.4±0.03 0.1±0.02 0.3±0.01
Vitamin C (%) 0.26±0.01 0.33±0.03 0.21±0.04 0.43±0.01 0.69±0.02 1.55±0.03
Moisture
(%)
19.2±1.0 18.19±2.0 20.4±1.0 19.2±3.0 19.2±2.0 21.5±3.0
Fats
(%)
0.3±0.02 0.5±0.03 0.7±0.01 0.6±0.03 0.5±0.01 0.4±0.04
pH
4.8±0.3 3.4±0.1 3.9±0.2 4.3±0.4 3.8±0.1 3.5±0.2
Acidity
(meq/kg)
32.2±2.0 33.1±3.0 35.3±1.0 37.2±2.0 36.1±3.0 43.3±1.0
Ash (%) 0.23±0.02 0.22±0.02 0.14±0.03 0.19±0.03 0.46±0.02 0.33±0.01
Electrical
conductivity
(mS/cm)
2.12±0.02 2.56±0.02 3.27±0.04 3.12±0.01 3.15±0.03 3.44±0.02
HMF
(mg/kg)
5.4±0.2 6.5±0.3 4.2±0.1 6.7±0.02 5.3±0.1 5.4±0.3
Reducing
sugar (%)
62.2±2.0 56.1±2.0 69.3±4.0 63.2±2.0 65.1±3.0 60.4±2.0
Sucrose
(%)
1.6±0.3 2.8±0.1 2.4±0.2 2.9±0.4 3.1±0.5 1.5±0.2
Total
84.59 78.34 93.05 87.63 89.15 85.89
* Mean ± S.D.
Maximum concentration (3.44±0.02) of electrical conductivity was found in Bekerr
honey, while minimum (2.12±0.02) in Big bee’s honey (Figure 4.6, o). In our samples,
263
maximum concentration of HMF (6.7±0.02) was found in Palosa honey, while minimum
(4.2±0.1) in Beera honey (Figure 4.6, r). Maximum concentration (69.3±4.0) of reducing
sugar was found in Beera honey, while minimum (56.1±2.0) in Small bee’s honey. Maximum
concentration (3.1±0.5) of sucrose was found in Sperkay honey, while minimum (1.5±0.2) in
Bekerr honey (Figure 4.6, n; 4.6, o).
Figure 4.6,di: Chemical composition of branded honey samples
264
Figure 4.6,d ii: Chemical composition of branded honey samples
Figure 4.6,e i: Chemical composition of unbranded honey samples
265
Figure 4.6,e ii: Chemical composition of unbranded honey samples
Figure 4.6,f i: Chemical composition of natural comb honey samples
266
Figure 4.6,f ii: Chemical composition of natural comb honey samples
Figure 4.6,g: pH concentration of branded honey samples
267
Figure 4.6,h: pH concentration of unbranded honey samples
Figure 4.6, I: pH concentration of natural honey samples
268
Figure4.6,j: Acidity concentration of branded honey samples
Figure4.6,k: Acidity concentration of unbranded honey samples
269
Figure 4.6,l: Acidity concentration of natural comb honey samples
Figure 4.6,m: Electrical conductivity concentration of branded honey samples
270
Figure 4.6,n: Electrical conductivity concentration of unbranded honey samples
Figure 4.6,o: Electrical conductivity concentration of natural comb honey samples
271
Figure 4.6,p: Hydroxy Methyl Furfural concentration in branded honey samples
Figure 4.6,q: Hydroxy Methyl Furfural concentration in unbranded honey samples
272
Figure 4.6,r: Hydroxy Methyl Furfural concentration in natural comb honey samples
273
4.7. Carbohydrates
According to the literature, honey has always been regarded as a food which is
advantageous for one’s health and as a product that has healing qualities. For this reason, it
is necessary to protect consumers from the fraudulent mislabeling of inferior honeys [394].
Karkacier et al., (2000) reported that sugar content of honeys varies according to the honey
type and the production region. For example, honeys from Italian Molise region in terms of
means showed 40.6 % fructose, 33.5 % glucose and 1.09 % sucrose to differentiate different
honeys from each other [395]. This study was focused to evaluate the concentration of
carbohydrates contents (g/100g) in branded, unbranded and natural comb honey of Khyber
Pakhtunkhwa Pakistan. HPLC chromatogram of carbohydrates standards is presented (Figure
4.7, a).
Figure4.7a: HPLC chromatogram of carbohydrates standard: 5.03 PMP, 8.02 α-D-
manose, 11.03 β-D-ribose, 13.45 β-D-lactose, 17.52 α-D-maltose, 23.01 α-D-
274
sucrose, 27.50 β-D-glucose, 31.50 α-D-xylose, 32.08 α-glactose, 36.52 α-D-
arabinose and 39.02 for β-D-fructose were identified.
In Table 4.44, branded honey samples show the maximum concentration (38.74±0.02) of β-
D-fructose were observed in Langnese honey, minimum (22.13±0.04) in Al-hayat honey,
whereas moderate (29.25±0.02) in Marhaba honey, versatile honey (34.41±0.03), Qarshi
(27.35±0.03), Pak-salman (26.83±0.02) and Young’s (23.34±0.03) respectively. Maximum
concentration (29.54±0.03) of β-D-glucose were observed in Langnese, minimum
(21.33±0.02) in Al-hayat honey, whereas moderate (24.63±0.04) in Marhaba honey, Young’s
honey (22.76±0.02), Pak-Salman (28.92±0.05), Versatile honey (26.43±0.03) and Qarshi
(24.82±0.03) respectively.
Antonini et al., (2006) reported that, values of fructose and glucose contents obtained in the
honey samples used in this study are in agreement with the previous works in the manner
that fructose always predominates [71]. Maximum concentrations (0.37±0.01) of α-D-
maltose were observed in Langnese honey, minimum (0.12±0.03) in Al-hayat honey,
whereas moderate in Marhaba (0.24±0.03) Young’s honey (0.22±0.04), Pak-Salman
(0.27±0.01), Versatile (0.33±0.02) and Qarshi (0.14±0.02) respectively.
Maximum concentration (0.15±0.01) of α-D-raffinose were observed in Al-hayat
honey, minimum (0.07±0.01) in Marhaba honey, whereas moderate (0.13±0.03) in Langnese
honey, Young’s honey (0.14±0.02), Pak-Salman (0.11±0.02), Versatile (0.09±0.02) and Qarshi
(0.10±0.01) respectively. Maximum concentration (16.92±0.03) of α-D-sucrose were
observed in Marhaba honey, minimum (8.64±0.04) in Langnese honey, whereas moderate
(11.22±0.01) in Qarshi honey, Young’s honey (15.12±0.04), Pak-salman (10.83±0.03),
Versatile (13.53±0.06) and Al-hayat (9.75±0.02) respectively (Figure 4.7, b i; 4.7, b ii).
275
Maximum concentration (0.08±0.02) of β-D-lactose were observed in Versatile honey,
minimum (0.02±0.02) in Marhaba honey, whereas moderate (0.03±0.02) in Langnese honey,
Young’s honey (0.03±0.01), Pak-salman (0.05±0.01), Qarshi (0.04±0.03) and Al-hayat
(0.06±0.03) respectively. Maximum concentration (0.17±0.02) of α-D-xylose were observed
in Versatile honey, minimum (0.07±0.01) in Langnese honey, whereas moderate (0.12±0.02)
in Marhaba honey, Young’s honey (0.13±0.03), Pak-salman (0.12±0.02), Qarshi (0.13±0.02)
and Al-hayat (0.15±0.03) respectively. Maximum concentration (0.11±0.01) of β-D-ribose
were observed in Langnese honey, minimum (0.04±0.01) in Pak-salman honey, whereas
moderate (0.07±0.02) in Marhaba honey, Young’s honey (0.09±02), Versatile (0.05±0.02),
Qarshi (0.08±0.01) and Al-hayat (0.07±0.02) respectively.
Maximum concentrations (0.73±0.04) of α-D-glactose were observed in Young’s
honey, minimum (0.33±0.04) in Marhaba honey, whereas moderate (0.36±0.02) in Pak-
salman, Langnese honey (0.37±0.02), Versatile (0.55±0.01), Qarshi (0.64±0.03) and Al-hayat
(0.49±0.03) respectively. Hogg et al., (1989) reported that Turkish honeys, sugar contents
and the profiles of different types of honeys were found variable. Pine and oak honeydews
were contained lower concentrations of fructose and glucose. 30.6-30.4g/100g for fructose
and 23.5 - 19.7 g/100g for glucose respectively. All the floral honeys were showed relatively
high concentrations of fructose (36.9 – 40.2 g/100g), with highest values being for cotton
and sunflower honeys which were highly granulated [69].
276
Table 4.44: Carbohydrates contents in branded honey (g/100g)
S.No Compounds Concentration of carbohydrates (g/100g)
Marhaba Versatile Qarshi Langnese Al-hayat Pak-salman Young’s
1 β-D-fructose 29.25±0.02 34.41±0.03 27.35±0.03 38.74±0.02 22.13±0.04 26.83±0.02 23.34±0.03
2 β-D-glucose 24.63±0.04 26.43±0.03 24.82±0.03 29.54±0.03 21.33±0.02 28.92±0.05 22.76±0.02
3 α-D-maltose 0.24±0.03 0.33±0.02 0.14±0.02 0.37±0.01 0.12±0.03 0.27±0.01 0.22±0.04
4 α-D-raffinose 0.07±0.01 0.09±0.02 0.10±0.01 0.13±0.03 0.15±0.01 0.11±0.02 0.14±0.02
5 α-D-sucrose 16.92±0.03 13.53±0.06 11.22±0.01 8.64±0.04 9.75±0.02 10.83±0.03 15.12±0.04
6 β-D-lactose 0.02±0.02 0.08±0.02 0.04±0.03 0.03±0.02 0.06±0.03 0.05±0.01 0.03±0.01
7 α-D-xylose 0.12±0.01 0.17±0.02 0.13±0.02 0.07±0.01 0.15±0.03 0.12±0.02 0.13±0.03
8 β-D-ribose 0.07±0.02 0.05±0.02 0.08±0.02 0.11±0.01 0.07±0.02 0.04±0.01 0.09±0.01
9 α-D-arabinos 0 0 0 0 0 0 0
10 α-D-glactose 0.33±0.04 0.55±0.01 0.64±0.03 0.37±0.02 0.49±0.03 0.36±0.02 0.73±0.04
Total 71 .65 75.64 64.52 78.00 54.25 67.53 62.56
In Table 4.45, unbranded honey shows the maximum concentration (31.57±0.03) of
β-D-fructose were observed in Beera honey, minimum (17.23±0.02) in Granda honey,
whereas moderate (23.45±0.02) in Big bee’s honey, Small bee’s honey(27.67±0.06), Bekker
(19.78±0.01) Sperkay (20.84±0.05) and Palosa (22.35±0.02) respectively. Maximum
concentration (38.66±0.02) of α-D-glactose was observed in Granda honey, minimum
(12.43±0.02) in Bekker honey, whereas moderate (16.91±0.04) in Sperkay honey, Small bee’s
honey (20.21±0.02), Palosa (26.77±0.03), Beera honey (34.72±0.03) and Big bee’s honey
(13.36±0.03) respectively. Corbella et al., (2006) reported that, when glucose is found in
higher concentrations, tends to crystallize spontaneously at room temperature in the form
277
of glucose monohydrate [70]. Maximum concentration (0.31±0.03) of α-D-maltose were
observed in Small bee’s honey while minimum (0.11±0.02) in Big bee’s honey, whereas
moderate (0.21±0.03) in Sperkay honey, Granda honey (0.19±0.05), Palosa (0.17±0.04),
Beera honey (0.26±0.04) and Bekker honey (0.29±0.02) respectively. Maximum
concentrations (0.14±0.01) of α-D-raffinose were observed in Palosa honey, minimum
(0.06±0.02) in Sperkay honey, whereas moderate (0.07±0.01) in Big bee’s honey, Granda
honey (0.09±0.01), Small bee’s honey (0.08±0.02), Beera honey (0.12±0.02) and Bekker
honey (0.03±0.01) respectively.
Table 4.45: Carbohydrates contents in unbranded honey (g/100g)
S.No Compounds Concentration of carbohydrates (g/100g)
Small bee’s
honey
Big bee’s
honey
Beera
Bekker
Palosa
Sperkay
Garranda
1 β-D-fructose 27.67±0.06 23.45±0.02 31.57±0.03 19.78±0.01 22.35±0.02 20.84±0.05 17.23±0.02
2 β-D-glucose 20.21±0.02 13.36±0.03 34.72±0.03 12.43±0.02 26.77±0.03 16.91±0.04 38.66±0.02
3 α-D-maltose 0.31±0.03 0.11±0.02 0.26±0.04 0.29±0.02 0.17±0.04 0.21±0.03 0.19±0.05
4 α-D-raffinose 0.08±0.02 0.07±0.01 0.12±0.02 0.03±0.01 0.14±0.01 0.06±0.02 0.09±0.01
5 α-D-sucrose 12.73±0.04 19.51±0.05 8.94±0.04 22.85±0.03 15.62±0.05 16.23±0.04 17.32±0.04
6 β-D-lactose 0.07±0.02 0.02±0.01 0.05±0.02 0.09±0.02 0.06±0.01 0.08±0.01 0.11±0.02
7 α-D-xylose 0.10±0.01 0.11±0.02 0.16±0.03 0.14±0.03 0.13±0.02 0.15±0.03 0.12±0.03
8 β-D-ribose 0.07±0.02 0.03±0.02 0.02±0.01 0.08±0.03 0.06±0.01 0.04±0.02 0.05±0.01
9 α-D-arabinos 0 0 0 0 0 0 0
10 α-D-glactose 0.31±0.03 0.21±0.04 0.26±0.02 0.22±0.03 0.38±0.02 0.32±0.03 0.17±0.03
Total 61.55 56.87 76.10 55.91 65.68 54.84 73.94
Maximum concentrations (22.85±0.03) of α-D-sucrose were observed in Bekker honey,
minimum (8.94±0.04) in Beera honey, whereas moderate (19.51±0.05) in Big bee’s honey,
Granda honey (17.32±0.04), Small bee’s honey (12.73±0.04), Palosa honey (15.62±0.05) and
278
Sperkay honey (16.23±0.04) respectively. Maximum concentrations (0.11±0.02) of β-D-
lactose were observed in Granda honey, minimum (0.02±0.01) in Big bee’s honey, whereas
moderate (0.08±0.01) in Sperkay honey, Beera honey (0.05±0.02), Small bee’s honey
(0.07±0.02), Palosa honey (0.06±0.01) and Bekker honey (0.09±0.02) respectively. Maximum
concentrations (0.16±0.03) of β-D-xylose were observed in Beera honey, minimum
(0.10±0.01) in Small bee’s honey, whereas moderate (0.15±0.03) in Sperkay honey, Granda
honey (0.12±0.02), Big bee’s honey (0.11±0.02), Palosa honey (0.13±0.02) and Bekker honey
(0.14±0.03) respectively. Maximum concentrations (0.08±0.03) of β-D-ribose were observed
in Bekker honey, minimum (0.02±0.01) in Beera honey, whereas moderate (0.04±0.02) in
Sperkay honey, Granda honey (0.05±0.01), Big bee’s honey (0.03±0.02), Palosa honey
(0.06±0.01) and Small bee’s honey (0.07±0.02) respectively. While α-D-arabinos was not
detected in any sample of natural and farm honey samples (Figure4.7, c I; 4.7, c ii).
In Table 4.46, natural combs honey shows that the maximum concentration
(41.61±0.01) of β-D-fructose was observed in Beera honey, minimum (33.33±0.03) in Big
bee’s honey, whereas moderate (38.32±0.02) in Sperkay, (40.24±0.02) Granda, (37.42±0.02)
Bekker and Palosa honey (39.53±0.03) respectively. Maximum concentrations (31.44±0.05)
of β-D-glucose were observed in Big bee’s honey, minimum (23.56±0.01) in Sperkay honey,
whereas moderate (29.34±0.03) in Small bee’s honey, Beera honey (27.23±0.01), Bekker
(25.51±0.03) Granda (26.67±0.02) and Palosa honey (27.42±0.04) respectively (Figure 4,7, d i
; 4.7, d ii).
Maximum concentrations (0.33±0.04) of α-D-maltose were observed in Beera
honey, minimum (0.19±0.02) in Sperkay honey, whereas moderate (0.24±0.05) in Small
bee’s honey, Big bee’s honey (0.31±0.06), Bekker (0.25±0.03) Granda (0.22±0.05) and Palosa
honey (0.29±0.03) respectively.
279
Maximum concentrations (0.23±0.06) of α-D-raffinose were observed in Beera honey,
minimum (0.09±0.04) in Palosa honey, whereas moderate (0.12±0.02) in Small bee’s honey,
Big bee’s honey (0.10±0.03), Bekker (0.18±0.03), Granda (0.15±0.03) and Sperkay honey
(0.20±0.03) respectively. Maximum concentrations (8.36±0.07) of α-D-sucrose were
observed in Bakker honey, minimum (3.44±0.05) in Big bee’s honey, whereas moderate
(5.92±0.07) in Small bee’s honey, Beera (6.90±0.02), Palosa (4.41±0.01), Granda (7.11±0.07)
and Sperkay honey (6.89±0.02) respectively.
Table 4.46: Carbohydrates contents in natural combs honey (g/100g)
Maximum concentrations (0.11±0.01) of β-D-lactose were observed in Sperkay honey,
minimum (0.05±0.01) in Small bee’s honey, whereas moderate (0.09±0.03) in Big bee’s
honey, Beera (0.06±0.02), Palosa (0.07±0.02), Granda (0.09±0.03) and Bakker honey
(0.10±0.01) respectively. Maximum concentrations (0.17±0.03) of α-D-xylose were observed
S.No Compounds Concentration of carbohydrates (g/100g)
Small bee’s
honey
Big bee’s
honey
Beera Bekker
Palosa
Sperkay
Garranda
1 β-D-fructose 40.18±0.04 33.33±0.03 41.61±0.01 37.42±0.02 39.53±0.03 38.32±0.02 40.24±0.02
2 β-D-glucose 29.34±0.03 31.44±0.05 27.23±0.01 25.51±0.03 27.42±0.04 23.56±0.01 26.67±0.02
3 α-D-maltose 0.24±0.05 0.31±0.06 0.33±0.04 0.25±0.03 0.29±0.03 0.19±0.02 0.22±0.05
4 α-D-raffinose 0.12±0.02 0.10±0.03 0.23±0.06 0.18±0.03 0.09±0.04 0.20±0.03 0.15±0.03
5 α-D-sucrose 5.92±0.07 3.44±0.05 6.90±0.02 8.36±0.07 4.41±0.01 6.89±0.02 7.11±0.07
6 β-D-lactose 0.05±0.01 0.09±0.03 0.06±0.02 0.10±0.01 0.07±0.02 0.11±0.01 0.09±0.03
7 α-D-xylose 0.08±0.02 0.11±0.02 0.16±0.03 0.17±0.03 0.07±0.02 0.12±0.02 0.13±0.01
8 β-D-ribose 0.06±0.02 0.07±0.01 0.14±0.02 0.08±0.01 0.13±0.02 0.10±0.01 0.05±0.02
9 α-D-arabinos 0 0 0 0 0 0 0
10 α-D-glactose 0.77±0.05 0.85±0.04 0.56±0.03 0.67±0.03 0.78±0.05 0.69±0.04 0.55±0.03
Total 76.76 69.74 77.22 72.74 72.79 70.18 75.21
280
in Bakker honey, minimum (0.07±0.02) in Palosa honey, whereas moderate (0.11±0.02) in
Big bee’s honey, Beera (0.16±0.03), Small bee’s honey (0.08±0.02), Granda (0.13±0.01) and
Sperkay honey (0.12±0.02) respectively.
Maximum concentrations (0.14±0.02) of β-D-ribose were observed in Beera honey,
minimum (0.05) in Granda honey, whereas moderate (0.06±0.02) in Small bee’s honey, Big
bee’s honey (0.07±0.01), Bakker (0.08±0.01) Palosa (0.13±0.02) and Sperkay honey
(0.10±0.01) respectively. Maximum concentrations (0.85±0.04) of α-D-glactose were
observed in Big bee’s honey, minimum (0.55±0.03) in Granda honey, whereas moderate
(0.77±0.05) in Small bee’s honey, Beera honey (0.56±0.03), Bekker (0.67±0.03), Palosa
(0.78±0.05) and Sperkay honey (0.69±0.04) respectively. Turkish honeys showed 34.29 %
fructose and 27.04 % glucose in the floral honeys and 37.49 % fructose and 31.55 % glucose
in the honeydew honeys [396].
Figure 4.7,b i: Concentration of major carbohydrates in branded honey samples
281
Figure 4.7,b ii: Concentration of minor carbohydrates in branded honey samples
Figure 4.7,c i: Concentration of major carbohydrates in unbranded honey samples
282
Figure 4.7,c ii: Concentration of minor carbohydrates in unbranded honey samples
Figure 4.7,d i: Concentration of major carbohydrates in natural honey samples
283
Figure 4.7,d ii: Concentration of minor carbohydrates in natural honey samples
4.8. Hydroxy Methyl Furfural
The aim of this study was to evaluate the effect of flame and oven heating on
Hydroxy Methyl Furfural content in natural and farms honey of Khyber Pakhtunkhwa. The
samples were collected and brought to PCSIR labs for investigation of increase of HMF
Quantity. (Table 4.47), shows Hydroxy Methyl Furfural (HMF) concentration (ppm) in farm
honey samples after treatment at different temperature (35, 50 and 70oC) for different time
period (20, 30, 40, 50 and 60 minutes). (Table 4.48), shows Hydroxy Methyl Furfural
concentration (ppm) in natural comb honey samples after treatment at different
temperature (35, 50, and 70oC) for different time period (20, 30, 40, 50 and 60 minutes).
(Table 4.49), Shows Hydroxy Methyl Furfural concentration (ppm) in farms honey
after flame heating at for different time periods (2, 5, 7, 9 and 12 minutes). (Table 4.50),
shows Hydroxy Methyl Furfural concentration (ppm) in natural comb honey after flame
heating for different time period (2, 5, 7, 9 and 12 minutes). The HMF contents increased in
all farm honey’s samples ranged from 100-159% kept for 60 minutes at 70oC oven. The HMF
contents increased in all natural honey’s samples ranged from 124-144% when kept for 60
284
minutes at 70oC in oven. The HMF contents increased in all farm honey’s samples ranged
from 407-593% for 12 minutes by flam heating. The HMF contents increased in all natural
honey’s samples ranged from 519-673% kept for 12 minutes by flam heating.
Hydroxy methyl furfural (HMF) considered is the most important derivative product
of heated honey reported by Turhan et al.,(2008) [397]. Initial concentration in farm honey,
Big bee’s honey was (7.12±0.01). After thermal treatment in electric oven for (20 and 30
minutes) duration at (35, 50 and 70oC) the concentration of (HMF) remains unchanged. The
maximum increase (159%) in (HMF) concentration was observed at 70oC for 60 minutes
treatments. Initial concentration in Small bee’s honey was (6.15±0.02). After thermal
treatment in electric oven for (20 and 30) minutes duration at (35, 50 and 70oC) the
concentration of (HMF) remain unchanged .Maximum increase (117%) in HMF concentration
was observed at 70oC for 60 minutes treatments. Initial concentration in Beera honey was
(8.23±0.01). After thermal treatment in electric oven for (20 and 30 minutes) duration at
(35, 50 and 70oC) the concentration of (HMF) remain unchanged .The maximum increase
(109 %) in HMF concentration was observed at 70oC for 60 minutes treatments (Figure 4.8,a
; 4.8,b and 4.8,c).
Initial concentration of (HMF) in Palosa honey was (5.31±0.02). After thermal
treatment in electric oven for (20 and 30 minutes) duration at (35, 50 and 70oC) the
concentration of (HMF) remains unchanged. The maximum increase (139%) in HMF
concentration was observed at 70oC for 60 minutes treatments. Initial concentration in
Bekerr honey was (6.40±0.03). After thermal treatment in electric oven for (20 and 30)
minutes duration at (35, 50 and 70oC) the concentration of (HMF) remain unchanged .the
maximum increase (100%) in HMF concentration was observed at 70oC for 60 minutes
treatments.
285
According to the council directive ANNEX II the HMF content of honey should be
lower than 40mg/kg. So it is the excellent indicator for the freshness of honey reported by
Hamdan et al., (2010) [85]. In the past it was reported by the Bath and Sing et al., (2001)
[174], that there is no significant change in the total acidity, pH, ash, glucose sucrose and
fructose content of honey were seen during heating Thrasyvoulou et al., (1986) observed
that the HMF concentration were increase from (0.0 to 8.8ppm) after one year storage
[398]. Fallico et al., (2004) reported that in Chestnut honey (HMF) contents were higher
than 40 mg kg−1 over 4 hours for 90°C and under 1 hour at 100°C [399]. Initial concentration
in natural comb honey, Big bee’s honey was (4.23±0.02). After thermal treatment in electric
oven for (20 and 30 minutes) duration at (35, 50 and 70°C) the concentration of (HMF)
remain unchanged. The maximum increase (125%) in HMF concentration was observed at
70°C for 60 minutes treatments. Initial concentration in Small bee’s honey was (6.34±0.03).
After thermal treatment in electric oven for (20 and 30 minutes) duration at (35, 50 and
70°C) the concentration of (HMF) remain unchanged. Maximum increase (133%) in HMF
concentration was observed at 70°C for 60 minutes treatments. Initial concentration in
Beera honey was (5.11±0.02). After thermal treatment in electric oven for (20 and 30
minutes) duration at (35, 50 and 70°C) the concentration of (HMF) remain unchanged. The
maximum increase (124%) in HMF concentration was observed at 70°C for 60 minutes
treatments (Figure 4.8, d; 4.8, e and 4.8, f).
Initial concentration of (HMF) in Palosa honey was (4.51±0.01). After thermal
treatment in electric oven for (20 and 30 minutes) duration at (35, 50 and 70°C) the
concentration of (HMF) remain unchanged. Maximum increase (118%) in (HMF)
concentration was observed at 70°C for 60 minutes treatments. Initial concentration in
natural comb honey, Bekerr honey was (5.27±0.02). After thermal treatment in electric oven
for (20 and 30 minutes) duration at (35, 50 and 70°C) the concentration of (HMF) remain
unchanged. Maximum increase (144%) in HMF concentration was observed at 70°C for 60
286
minutes treatments (Figure 4.8, g; 4.8, h). Hartel et al., (1991) reported that , For Lime
honey, HMF concentrations were found higher than 40 mg kg−1 over 24 hours for 70 °C,
over 1 hour at 90 °C and under 1 hour at 100°C [400]. Initial concentration of (HMF) in Farm
honey, Big bee’s honey was (7.12±0.01). After flame heating the maximum increase (503 %)
in HMF concentration was observed at 12 minutes treatments. Initial concentration in Small
bee’s honey was (6.15±0.02). After flam heating the maximum increase (442 %) in HMF
concentration was observed at 12 minutes treatments. Initial concentration in Beera honey
was (8.23±0.01). After flame heating the maximum increase (407%) in HMF concentration
was observed at 12 minutes treatments. Initial concentration in Palosa honey was
(5.31±0.02). After flame heating the maximum increase (593 %) in HMF concentration was
observed at 12 minutes treatments. Initial concentration in Bekerr honey was (6.40±0.03).
After flame heating the maximum increase (539.0%) in HMF concentration was observed at
12 minutes treatments (Figure4.8, i).
Skinner et al., (2009) reported that at 75oC HMF is extremely high except in pine
honey which just exceeded 40 mg/kg. Helianthus honey HMF concentrations were increased
from 26 to 226.80 mg/kg. In cotton honey HMF were increase from 9 to 63 mg/kg and in
thymus honey the increase were observed from 8 to 191 mg/kg respectively [89]. Initial
concentration in natural comb honeys, Big bee’s honey was (4.23±0.02). After flame
heating the maximum increase (592%) in HMF concentration was observed at 12 minutes
treatments. Initial concentration in Small bee’s honey was (6.34±0.03). After flame heating
the maximum increase (627%) in HMF concentration was observed at 12 minutes
treatments. Initial concentration in Beera honey was (5.11±0.02). After flame heating the
maximum increase (519 %) in HMF concentration was observed at 12 minutes treatments.
Initial concentration in Palosa honey was (4.51±0.01).
287
Table 4.47: Effect of temperature on H.M.F concentration in farms honey
N/S
Heating
time
(minutes)
HMF initial
concentration
(ppm)
HMF concentration at different
Temperature (ºC)
Increase
(%)
35 50 70
Big
bee’s
honey
20
7.12±0.01
7.12±0.01 7.12±0.01 7.12±0.02 0.00
30 7.12±0.01 7.12±0.02 7.12±0.01 0.00
40 7.12±0.01 7.13±0.01 7.14±0.01 2.00
50 7.31±0.02 7.78±0.01 7.99±0.02 78.00
60 8.34±0.02 8.51±0.01 8.71±0.03 159.00
Small
bee’s
honey
20
6.15±0.02
6.15±0.01 6.15±0.02 6.15±0.03 0.00
30 6.15±0.01 6.15±0.03 6.15±0.01 0.00
40 6.15±0.02 6.16±0.01 6.17±0.01 3.00
50 6.23±0.02 6.48±0.01 6.61±0.01 46.00
60 6.83±0.02 7.14±0.02 7.32±0.03 117.00
Beera
20
8.23±0.01
8.23±0.02 8.23±0.03 8.23±0.03 0.00
30 8.23±0.01 8.23±0.02 8.23±0.02 0.00
40 8.23±0.02 8.24±0.02 8.25±0.02 2.00
50 8.35±0.01 8.46±0.02 8.69±0.01 46.00
60 8.99±0.02 9.19±0.01 9.32±0.02 109.00
Palosa 20
5.31±0.02
5.31±0.01 5.31±0.03 5.31±0.02 0.00
30 5.31±0.02 5.31±0.01 5.31±0.01 0.00
40 5.31±0.02 5.32±0.02 5.33±0.03 2.00
50 5.40±0.02 5.67±0.03 5.97±0.01 66.00
60 6.29±0.01 6.56±0.02 6.70±0.02 139.00
Bekerr 20
6.40±0.02 6.40±0.02 6.40±0.01 0.00
30 6.40±0.01 6.40±0.02 6.40±0.02 0.00
288
40 6.40±0.03 6.40±0.01 6.41±0.02 6.42±0.01 2.00
50 6.55±0.02 6.72±0.01 6.89±0.03 48.00
60 6.99±0.02 7.23±0.03 7.40±0.02 100.00
N/S= Name of Samples
Table 4.48: Effect of temperature on H.M.F concentration in natural comb honey
N/S
Heating
time
(minutes)
HMF initial
concentration
(ppm)
HMF concentration at different
Temperature (ºC)
Increase
(%)
35 50 70
Big
bee’s
honey
20
4.23±0.02
4.23±0.01 4.23±0.02 4.23±0.01 0.00
30 4.23±0.03 4.23±0.01 4.23±0.02 0.00
40 4.24±0.01 4.25±0.02 4.26±0.01 2.00
50 4.31±0.02 4.56±0.01 4.73±0.02 50.00
60 4.95±0.02 5.29±0.01 5.48±0.02 125.00
Small
bee’s
honey
20
6.34±0.03
6.34±0.02 6.34±0.01 6.34±0.02 0.00
30 6.34±0.01 6.34±0.01 6.34±0.02 0.00
40 6.35±0.01 6.36±0.03 6.37±0.01 3.00
50 6.48±0.02 6.67±0.02 6.88±0.02 54.00
60 7.13±0.01 7.31±0.02 7.67±0.02 133.00
Beera
20
5.11±0.02
5.11±0.01 5.11±0.03 5.11±0.01 0.00
30 5.11±0.01 5.11±0.02 5.11±0.03 0.00
40 5.12±0.03 5.14±0.01 5.15±0.02 4.00
50 5.19±0.01 5.37±0.02 5.62±0.01 50.00
60 5.81±0.01 6.02±0.02 6.35±0.01 124.00
Palosa
20
4.51±0.01
4.51±0.03 4.51±0.01 4.51±0.02 0.00
30 4.51±0.01 4.51±0.02 4.51±0.03 0.00
40 4.51±0.02 4.52±0.01 4.53±0.01 2.00
50 4.63±0.01 4.86±0.02 5.06±0.01 55.00
289
60 5.37±0.01 5.42±0.02 5.69±0.01 118.00
Bekerr 20
5.27±0.02
5.27±0.01 5.27±0.01 5.27±0.02 0.00
30 5.27±0.01 5.27±0.02 5.27±0.03 0.00
40 5.27±0.02 5.28±0.02 5.29±0.02 2.00
50 5.42±0.02 5.68±0.02 5.94±0.02 67.00
60 6.26±0.02 6.47±0.01 6.71±0.02 144.00
N/S= Name of Samples
Table 4.49: Effect of flame heating on H.M.F concentration in farms honey
N/S
Heating
Time
(minutes)
HMF initial concentration
(ppm)
HMF concentration after
heating
Increase
(%)
Big
bee’s
honey
2
7.12±0.01
7.44±0.02 32.00
5 7.98±0.01 86.00
7 8.33±0.01 121.0
9 9.65±0.01 253.0
12 12.15±0.03 503.0
Small
bee’s
honey
2
6.15±0.02
6.22±0.03 07.00
5 6.60±0.01 44.90
7 7.25±0.01 110.0
9 8.24±0.01 209.0
12 10.57±0.03 442.0
Beera
2
8.23±0.01
8.42±0.03 19.00
5 8.94±0.02 70.99
7 9.35±0.03 112.0
9 10.97±0.01 274.0
12 12.30±0.03 407.0
2 5.55±0.02 24.00
290
Palosa 5
5.31±0.02
5.91±0.01 60.00
7 6.55±0.03 124.0
9 9.00±0.02 369.0
12 14.24±0.01 593.0
Bekerr 2
6.40±0.03
6.54±0.01 14.00
5 7.67±0.02 127.0
7 8.99±0.01 259.0
9 9.23±0.03 283.0
12 11.79±0.03 539.0
N/S= Name of Samples
Table 4.50: Effect of flame heating on H.M.F concentration in natural comb honey
N/S
Heating
Time(min)
HMF initial
concentration (ppm)
HMF concentration after
heating
Increase
(%)
Big
bee’s
honey
2
4.23±0.02
4.34±0.01 10.99
5 4.88±0.02 65.00
7 5.40±0.01 117.0
9 7.65±0.01 342.0
12 10.15±0.03 592.0
Small
bee’s
honey
2
6.34±0.03
6.45±0.03 11.00
5 6.92±0.02 58.00
7 7.50±0.01 116.0
9 9.44±0.02 310.0
12 12.61±0.02 627.0
Beera
2
5.38±0.01 27.00
5 5.89±0.03 78.00
291
7 5.11±0.02 6.29±0.01 118.0
9 8.97±0.01 386.0
12 10.30±0.02 519.0
Palosa
2
4.51±0.01
4.69±0.01 18.00
5 5.08±0.02 57.00
7 6.70±0.03 219.0
9 8.00±0.01 349.0
12 11.24±0.02 673.0
Bekerr 2
5.27±0.02
5.47±0.03 20.00
5 6.11±0.01 84.00
7 7.47±0.02 220.0
9 9.19±0.01 392.0
12 10.96±0.02 569.0
N/S= Name of Samples
Aminov et al., (2010) noticed that HMF increased slowly until 70ºC, and then a sharp
increase was observed at higher temperatures [94]. According to the Silver et al., (2001)
observation HMF increased from 10.1ppm to 32.8ppm by heating of honey for 1 min [95].
After flame heating the maximum increase (673%) in HMF concentration was observed at 12
minutes treatments. Initial concentration in Bekerr honey was (5.27±0.02) (Figure 4.8, j).
After flame heating the maximum increase (569%) in HMF concentration was observed at 12
minutes treatments. In cotton honey, the HMF content increased obviously from 6.10ppm
for unheated sample to 6.22, 6.88, 9.70 and 16.90ppm by heating for 5 min, at 60ºC, 70ºC,
80ºC and 90ºC respectively.
292
Figure 4.8,a: Initial concentration of Hydroxy Methyl Furfural in farm honey samples
Figure 4.8,b: Initial concentration of Hydroxy Methyl Furfural in natural honey samples
293
Figure 4.8,c: Effect of temprature (35ºC) on Hydroxy Methyl Furfural concentration in
farm honey samples
Figure 4.8,d: Effect of temprature (50ºC) on Hydroxy Methyl Furfural concentration in farm
honey samples
294
Figure 4.8,e: Effect of temprature (70 ºC) on Hydroxy Methyl Furfural concentration in farm
honey samples
Figure 4.8,f: Effect of temprature (35 ºC) on Hydroxy Methyl Furfural concentration in
natural honey samples
295
Figure 4.8,g: Effect of temprature (50ºC) on Hydroxy Methyl Furfural concentration in
natural honey samples
Figure 4.8,h: Effect of temprature (70ºC) on Hydroxy Methyl Furfural concentration in
natural honey samples
296
Figure 4.8,i: Effect of flame heating on Hydroxy Methyl Furfural concentration in farm
honey samples
Figure 4.8,j: Effect of flame heating on Hydroxy Methyl Furfural concentration in natural
comb honey samples
4.9. Contaminants
297
This study presents the quantitative evaluation of aflatoxins (B1, B2, G1 and G2) and
heavy metals (cadmium, manganese, lead, mercury, nickel, cobalt and cupper) in branded,
unbranded and natural comb honey samples. (Table 4.51), presents heavy metals
concentration in branded honey samples, which showed that in Marhaba, nickel
concentration (0.49±0.03) found maximum and cobalt (0.15±0.02) was lowest, while
manganese (0.23±0.03), mercury (0.21±0.01) cadmium (0.17±0.02), cupper (0.16±0.03) and
lead (0.16±0.01) were in moderate concentration. Tuzen et al., (2007) reported that, lead
and cadmium contents in honeys were (8.4-105) ppb and (0.9 - 17.9) respectively [218]. In
Qarshi honey samples Lead concentration (0.85±0.03) found maximum and cadmium
(0.16±0.03) found lowest, while nickel (0.52±0.02), cupper (0.42±0.01), manganese
(0.34±0.02), cobalt (0.27±0.03) and mercury (0.25±0.02) were in moderate concentration.
Versatile contains maximum lead (1.34±0.02) while lowest cadmium (0.12±0.02), while
nickel (1.13±0.03), cupper (0.35±0.02), mercury (0.24±0.01), manganese (0.17±0.02) and
cobalt (0.13±0.01) were in moderate concentration.
Table 4.51: Heavy metals concentration in branded honey (µg/kg)
Honey
Samples
Cd Cu Pb Ni Mn Co Hg
Marhaba 0.17±0.02 0.16±0.03 0.16±0.01 0.49±0.03 0.23±0.03 0.15±0.02 0.21±0.01
Qarshi 0.16±0.03 0.42±0.01 0.85±0.03 0.52±0.02 0.34±0.02 0.27±0.03 0.25±0.02
Versatile 0.12±0.02 0.35±0.02 1.34±0.02 1.13±0.03 0.17±0.02 0.13±0.01 0.24±0.01
Al-hayat 0.18±0.02 1.23±0.03 0.11±0.03 0.44±0.02 0.12±0.02 0.19±0.01 0.28±0.02
Young’s
honey
0.20±0.03 0.77±0.03 1.03±0.03 2.41±0.01 0.24±0.03 0.18±0.02 0.16±0.03
Pak-salman 0.22±0.01 1.19±0.01 0.27±0.02 1.25±0.02 0.14±0.03 0.22±0.02 0.44±0.02
Langnese 0.13±0.02 0.47±0.03 0.17±0.03 0.13±0.01 0.18±0.03 0.14±0.03 0.71±0.03
± Mean Standard Deviation
298
Table 4.52: Heavy metals concentration in unbranded honey (µg/kg)
Honey Samples Cd Cu Pb Ni Mn Co Hg
Big bee’s honey 0.15±0.03 0.36±0.01 0.73±0.03 0.33±0.01 0.63±0.02 0.16±0.02 0.18±0.03
Small bee’s
honey
0.13±0.03 0.32±0.01 1.25±0.03 0.15±0.02 0.14±0.02 0.17±0.01 0.27±0.03
Beera 0.23±0.02 1.29±0.02 0.43±0.02 0.61±0.03 0.19±0.03 0.21±0.03 0.61±0.01
Palosa 0.11±0.02 0.13±0.01 1.27±0.02 0.19±0.03 0.26±0.01 0.65±0.01 0.55±0.02
Sperkay 0.14±0.02 0.41±0.03 0.39±0.03 0.94±0.03 1.15±0.02 0.46±0.02 0.69±0.01
Bekerr 0.17±0.01 0.12±0.03 0.16±0.03 0.46±0.02 0.32±0.03 0.17±0.02 0.13±0.03
Granda 0.24±0.03 0.15±0.02 0.52±0.02 2.25±0.01 0.15±0.03 0.25±0.03 0.12±0.02
± Mean Standard Deviation
Table 4.53: Heavy metals concentration in natural comb honey (µg/kg)
Honey Samples Cd Cu Pb Ni Mn Co Hg
Big bee’s honey ND 0.13±0.03 ND ND ND 0.12±0.03 ND
Small bee’s honey ND 0.12±0.02 ND 0.10±0.01 ND ND ND
Beera 0.13±0.01 ND ND 0.21±0.02 ND ND ND
Palosa ND ND 0.15±0.01 ND ND ND ND
Sperkay ND ND ND 0.14±0.01 ND 0.25±0.03 ND
Bekerr ND 0.12±0.01 0.12±0.02 ND 0.24±0.01 ND ND
Granda 0.15±0.01 ND ND 0.33±0.03 ND 0.13±0.02 ND
299
± Mean Standard Deviation ND: Not Detected
Table 4.54: Mycotoxins concentration in branded honey (µg/kg)
Honey Samples B1 B2 G1 G2 Total
Marhaba ND ND ND ND ND
Qarshi ND ND ND ND ND
Versatile ND ND ND ND ND
Al-hayat 1.25 ND ND ND 1.25
Young’s honey ND 2.14 ND ND 2.14
Pak-salman ND ND ND ND ND
Langnese ND ND ND ND ND
ND= Not detected
300
Table 4.55: Mycotoxins concentration in unbranded honey (µg/kg)
Honey Samples B1 B2 G1 G2 Total
Big bee’s honey ND ND ND ND ND
Small bee’s honey ND ND ND ND ND
Beera ND ND ND ND ND
Palosa ND ND ND ND ND
Sperkay ND 2.15 ND ND 2.15
Bekerr 2.33 ND ND ND 2.33
Granda ND ND ND ND ND
ND= Not Detected
Table 4.56: Mycotoxins concentration in natural comb honey (µg/kg)
Honey Samples B1 B2 G1 G2 Total
Big bee’s honey ND ND ND ND ND
Small bee’s honey ND ND ND ND ND
Beera ND ND ND ND ND
Palosa ND ND ND ND ND
Sperkay ND ND ND ND ND
Bekerr ND ND ND ND ND
Granda ND ND ND ND ND
ND= Not Detected
301
On the other hand, according to World Health Organization the average recommended daily
intake of Cd and Pb are 60μg/d and 210μg/d respectively. Therefore, consuming 20 g per
day of honey from Sanlıurfa provides: 0.06 μg Cd day and 6.98 µg Pb in a day [318]. In Al-
hayat, cupper concentration (1.23±0.03) found maximum and lead (0.11±0.03) was lowest,
while nickel (0.44±0.02), mercury (0.28±0.02), Cobalt (0.19±0.01), cadmium (0.18±0.02) and
manganese (0.12±0.02) were in moderate concentration. Young’s honey contains maximum
nickel (2.41±0.01) and lowest mercury (0.16±0.03), while lead (1.03±0.03), cupper
(0.77±0.03), manganese (0.24±0.03), cadmium (0.20±0.03) and cobalt (0.18±0.02) were in
moderate concentration. Yilmiz et al., (1999) reported that manganese level was 1.0 ppm in
honey obtained from different areas of southeastern Anatolia [319].
But in our samples the nickel concentration (1.25±0.02) found maximum and
manganese (0.14±0.03) lowest in Pak-salman, while cupper (1.19±0.01), mercury
(0.44±0.02), lead (0.27±0.02), cobalt (0.22±0.02) and cadmium (0.22±0.01) were in moderate
concentration. Mercury level in various bee’s products were reported as (0.00001 – 0.006
mg / Kg) [401]. In Langnese honey samples mercury concentration (0.71±0.03) found
maximum and cadmium (0.13±0.02) was lowest, while cupper (0.47±0.03), manganese
(0.18±0.03), lead (0.17±0.03) cobalt (0.14±0.03) and nickel (0.13±0.01) were in moderate
concentration (Figure 4.9, a). Conti et al., (2001) reported that around the world, cadmium
contents of some honey samples were (0.020-0.490 mg/kg) [220]. (Table 4.52), represents
heavy metals concentration in unbranded honey samples, which showed that in Big bees
honey, lead concentration (0.73±0.03) found maximum and cadmium (0.15±0.03) was
lowest, while manganese (0.63±0.02), cupper (0.36±0.01), nickel (0.33±0.01), mercury
(0.18±0.03) and cobalt (0.16±0.02) were in moderate concentration. Lead concentration
(1.25±0.03) found maximum and cadmium (0.13±0.03) lowest in small bee’s honey, while
302
cupper (0.32±0.01), mercury (0.27±0.03), cobalt (0.17±0.01), nickel (0.15±0.02) and
manganese (0.14±0.02) were in moderate concentration. It has been reported that the
manganese concentrations of Turkish honey were 0.31 ppm respectively [402]. Beera
contains maximum cupper (1.29±0.02) and lowest manganese (0.19±0.03), while mercury
(0.61±0.01) nickel (0.61±0.03), lead (0.43±0.02), cadmium (0.23±0.02) and cobalt (0.21±0.03)
were in moderate concentration. In Palosa, lead concentration (1.27±0.02) found maximum
and cadmium (0.11±0.02) was lowest, while cobalt (0.65±0.01), mercury (0.55±0.02),
manganese (0.26±0.01), nickel (0.19±0.03, cupper (0.13±0.01) were in moderate
concentration. It has been reported that manganese level of honey was (0.49) ppm [320].
Sperkay honey contains maximum manganese (1.15±0.02) and cadmium (0.14±0.02)
was lowest, while nickel (0.94±0.03), mercury (0.69±0.01), cobalt (0.46±0.02), cupper
(0.41±0.03) and lead (0.39±0.03) were in moderate concentration. Mercury concentration
(0.46±0.03) found maximum and cupper (0.46±0.03) was lowest in Bekerr, while nickel
(0.46±0.02), manganese (0.32±0.03), cobalt (0.17±0.02), cadmium (0.17±0.01) and lead
(0.16±0.03) were in moderate concentration. In Granda honey, nickel concentration
(2.25±0.01) found maximum and mercury (0.12±0.02) was lowest, while lead (0.52±0.02),
cobalt (0.25±0.03), cadmium (0.24±0.03), cupper (0.15±0.02) and manganese (0.15±0.03)
were in moderate concentration (Figure 4.9, b). Ni levels (0.004 – 3.23 mg/kg) have been
reported by Porrini et al., (2002) in honey under the Swiss MRL study [403]. Van et al., 1991)
reported that Lead is another heavy metal; their higher concentration leads to brain
defection, hypertension, hearing difficulty, anemia, and kidney disease and loses of
intelligence [404]. According to Frias et al., (2008) the average recommended daily intake of
Cd and Pb are 60μg/d and 210μg/d, respectively [322].
(Table 4.53), represents heavy metals concentration in natural comb honey samples, which
showed that in Big bee’s honey, cupper concentration (0.13±0.03) found maximum and
303
cobalt (0.12±0.03) was lowest, while manganese, nickel, mercury, cadmium and lead were
not detected. Cupper concentration (0.12±0.02) found maximum and nickel (0.10±0.01)
lowest in Small bee’s honey, while, lead, mercury, cobalt, cadmium and manganese were
not detected. Beera contains maximum nickel (0.21±0.02) and lowest cadmium (0.13±0.01),
while mercury, lead, manganese, copper and cobalt were not detected. In Palosa, lead
concentration (0.15±0.01) found maximum and (0.11±0.02), while cadmium, cobalt,
mercury, manganese, nickel and copper was not detected.
Sperkay honey contains maximum cobalt (0.25±0.03) and nickel (0.14±0.01) was
lowest, while cadmium, mercury, manganese, copper and lead were not detected.
Manganese concentration (0.24±0.01) found maximum, lead and cupper (0.12±0.01,
0.12±0.02) was lowest in Bekerr, while nickel, mercury, cobalt and cadmium were not
detected. In Granda honey, nickel concentration (0.33±0.03) found maximum. Cadmium
(0.15±0.01) was lowest, while cobalt (0.13±0.02) was in moderate concentration and lead,
mercury, cupper and manganese were not detected (Figure 4.9, c).
Detection of aflatoxins level in branded and unbranded honey has been reported
(Table 4.54 and 4.55). It showed that aflatoxins were not detected mostly in honey samples.
Higher concentration of aflatoxins B2 was found in Young’s honey (2.14 ppb), and lowest in
Al-hayat honey (1.25 ppb). Higher concentration of aflatoxin B1 (2.33 ppb) in Bekerr honey,
while the lower concentration of aflatoxin B2 were detected in Sperkay (2.15ppb) (Figure
4.9, d; 4.9, e). Detection of aflatoxins level in natural comb honey has been reported in Table
4.56. It showed that aflatoxins were not detected in any natural comb collected honey
samples.
304
Figure 4.9,a: Concentration of heavy metals in branded honey samples
Figure 4.9,b: Concentration of heavy metals in unbranded honey samples
305
Figure 4.9,c: Concentration of heavy metals in natural comb honey samples
Figure 4.9,d: Concentration of mycotoxin in branded honey samples
306
Figure 4.9,e: Concentration of mycotoxin in unbranded honey samples
CONCLUSIONS
Branded, unbranded and natural honey samples were evaluated for antibiotic
residues and their metabolites such as tetracycline, streptomycin, gentamycin,
penicillin, sulfonamide, chloramphenicol and nitrofuran. The results showed that
penicillin, streptomycin and oxytetracycline residue was found maximum in
unbranded honey as compare to branded honey, while not detected in natural
honey samples. Whereas gentamycin, sulfonamide, chloramphenicol, nitrofuran
and their metabolites were not detected in any sample. So it is concluded from
our study that streptomycin and oxytetracycline were extensively used by the
bee keepers of Khyber Pakhtunkhwa Pakistan for curing the diseases in bees.
All the branded, unbranded and natural honey samples evaluated showed
antioxidant activity. Natural comb honey samples presented better activity as
compared to farm honey samples.
307
Most of the honey’s samples contain phenolic acids such as chloroganic acid,
gallic acid, vanallic acid, benzoic acid and syringic acid. Natural combs honey
presented higher concentration of phenolic acids as compared to branded and
unbranded honey’s sample.
Result showed that undiluted honey have able to inhibit more growth of E. coli,
Bacillus cereus and Aspergillus niger but there was no effict on Candida
albicans. The honey of khyber Pukhtunkhwa has effective inhibitory affects
and have antimicrobial activity.
All these verities contains nutrients especially as energy provider sugar, vitamin
C and phenolic compounds which having medicinal importance. In natural
comb honey the concentration and quantity of ash, pH, moisture, total acidity,
electrical conductivity and total sugars contents are better as compare to branded
and unbranded honey.
All natural, branded and unbranded honey’s samples also contains
carbohydrates contents which are important for everyday life, biological
functions such as providing energy for running vital roles of the living body.
The Hydroxy Methyl Furfural concentrations were not much increase in electric
oven heating. At high temperature up to 70℃ the Hydroxy Methyl Furfural
concentration were increased.
Some branded and unbranded (farms) honey samples from local markets of
Khyber Pakhtunkhwa are contaminated with aflatoxins, while most of the
samples contain toxic heavy metals contamination in branded and unbranded
honey. The contaminated samples have lower concentration than the permissible
limits set by European commission and WHO. The aflatoxins analysis revealed
mainly the presence of aflatoxins B1 and B2, which shows the possibility of
308
fungal contamination during their production, marketing and storage. The toxic
metals contamination in honey may be from environment, while in natural comb
honey the concentration of heavy metal were in traces and aflatoxins were not
detected in any sample.
Thus specifically honey may constitute a suitable source and could be used as
alternative natural antioxidant in different formulations for the preparation of
food and pharmaceutical products, which is very well evidenced by the present
work. This study proved that honey of Khyber Pakhtunkhwa has the potential
for the therapeutic use.
The electric oven can be utilized as a processing tool for processing of honey
and to avoid direct flame heating of honey because it increases the HMF
concentration more as compare to oven heating. These honeys can be utilized as
good source of macronutrients in food as well as in herbal products. So these
sources of honey may be utilized as such, in different food; in turn will enhance
the export.
309
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