STUDY OF RESIDUAL ANTIBIOTICS AND THEIR METABOLITES IN …

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


3.9.2. 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


3.12.2. 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

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


UNBRANDED

HONEY (MG/KG)

126


NATURAL HONEY (MG/KG)

126

18

4.5 CONCENTRATION OF SULFONAMIDE ANTIBIOTIC IN

BRANDED HONEY SAMPLES

130


UNBRANDED HONEY SAMPLES

131


NATURAL HONEY SAMPLES

131

4.8 CONCENTRATION OF CHLORAMPHENICOL

ANTIBIOTIC RESIDUES IN BRANDED HONEY SAMPLES

132


ANTIBIOTIC RESIDUES IN UNBRANDED HONEY

SAMPLES

133


ANTIBIOTIC RESIDUES IN NATURAL HONEY SAMPLES

133

4.11 CONCENTRATION OF NITROFURAN AND THEIR

METABOLITES IN BRANDED HONEY SAMPLES

146


METABOLITES IN UNBRANDED HONEY SAMPLES

147

19


METABOLITES IN NATURAL HONEY SAMPLES

147

4.14 DPPH RADICAL SCAVENGING ACTIVITY OF


151



152


NATURAL COMB HONEY SAMPLES

152


BRANDED HONEY

(EC50 IN µG/G)

152


UNBRANDED HONEY

(EC50 IN µG/G)

153


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


180

4.37 QUALITATIVE TEST FOR PHYTOCHEMICALS IN


180

4.38 QUANTITATIVE TEST FOR PHYTOCHEMICALS IN


182



182



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


128

4.1E CONCENTRATION OF ANTIBIOTIC RESIDUES IN


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







OXA- ZOLIDINONE



OXA- ZOLIDINONE



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


158

4.4C CONCENTRATION OF PHENOLIC ACID IN


158

4.4D CONCENTRATION OF PHENOLIC ACID IN


159

4.5A ANTIFUNGAL ACTIVITY OF BRANDED HONEY

SAMPLES AGAINST ASPERGILLUSNIGER

167

4.5B ANTIFUNGAL ACTIVITY OF UNBRANDED HONEY


167

4.5C ANTIFUNGAL ACTIVITY OF NATURAL COMB HONEY


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



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


NATURAL COMB HONEY SAMPLES AGAINST E.COLI



BACILLUS CEREUS

177

177

4.6A CONCENTRATION OF PHYTOCHEMICALS IN


183

4.6B CONCENTRATION OF PHYTOCHEMICALS IN


184

4.6C CONCENTRATION OF PHYTOCHEMICALS IN


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


196

4.6N ELECTRICAL CONDUCTIVITY CONCENTRATION OF


196

4.6O ELECTRICAL CONDUCTIVITY CONCENTRATION OF


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


206

4.7B (II) CONCENTRATION OF MINOR CARBOHYDRATES IN


207

4.7C (I) CONCENTRATION OF MAJOR CARBOHYDRATES IN


207

4.7C (II) CONCENTRATION OF MINOR CARBOHYDRATES IN


208

4.7D (I) CONCENTRATION OF MAJOR CARBOHYDRATES IN


208

4.7D (II) CONCENTRATION OF MINOR CARBOHYDRATES IN


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


HONEY SAMPLES

220

4.8E EFFECT OF TEMPRATURE (70°C) ON HYDROXY


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


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

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

http://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].

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-oxazolidinone

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

http://jn.nutrition.org/content/134/12/3479S/F1.large.jpg

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

116

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].

118

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

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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

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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

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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

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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

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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

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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].

137

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].

138

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)

152

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

158

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.


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.


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.


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.


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


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


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

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)


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




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


195

ND: Not Detected



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-oxazolidinone;

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-oxazolidinone

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-


208

Figure 4.2,k: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-


209

Figure 4.2,l: LCMS-MS chromatogram of Furaltadone AMOZ = 3-amino-5-morpholino-

methyl-1, 3-oxazolidinone 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)


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


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


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


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


Table 4.22: Phenolic acids contents of natural combs honey samples


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


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




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


Strains Code Concentration Big

bees

honey

Small

bees

honey

Beera

Palosa

Sperkay

Bekerr

Granda

C. albicans

ATCC

90028




230



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)



bees

honey

Small

bees

honey

Beera

Palosa

Sperkay

Bekerr

Granda

Candida

albicans

ATCC

90028






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




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)




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


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


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)


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)



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)



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)



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

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).

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)


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


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


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


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


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


Figure 4.8,h: Effect of temprature (70ºC) on Hydroxy Methyl Furfural concentration in


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)


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)


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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