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UGWUONA, Fabian Uchenna
(PG/Ph.D/11/61287)
PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF FOUR NIGERIAN
SPICES
FACULTY OF AGRICULTURE
DEPARTMENT OF FOOD SCIENCE & TECHNOLOGY
Paul Okeke
Digitally Signed by: Content manager’s Name DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre
ii
PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF FOUR
NIGERIAN SPICES
By
UGWUONA, Fabian Uchenna (PG/Ph.D/11/61287)
DEPARTMENT OF FOOD SCIENCE & TECHNOLOGY FACULTY OF AGRICULTURE
UNIVERSITY OF NIGERIA, NSUKKA
JUNE, 2014
3
PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF FOUR NIGERIAN
SPICES
A Thesis Submitted to the Department of Food Science & Technology, Faculty of Agriculture,
University of Nigeria, Nsukka
In Partial Fulfillment of the Requirements for the Award of the Degree of Doctor of Philosophy in Food Science and Technology,
University of Nigeria, Nsukka
BY
Ugwuona, Fabian Uchenna PG/Ph. D/11/61287
Department of Food Science & Technology, Faculty of Agriculture,
University of Nigeria, Nsukka.
JUNE, 2014.
4
APPROVAL PAGE
This thesis has been approved for the award of the degree of Doctor of Philosophy in Food
Science and Technology, University of Nigeria, Nsukka.
By
………………………. …………………………… Prof. (MRS) J. C. ANI Thesis Supervisor External Examiner ……………………… …………………………….. DR. P.O. UVERE Head of Department Dean of Faculty
5
CERTIFICATION
Ugwuona, Fabian Uchenna, a postgraduate student in the Department of Food Science
and Technology, Faculty of Agriculture, University of Nigeria, Nsukka, has satisfactorily
completed the requirements for the degree of Doctor of Philosophy (Ph. D) in Food Science
and Technology. The work embodied in this thesis is original and has not been submitted in
part or full for any other diploma or degree of this or any other university.
………………………………… Prof. (MRS.) J. C. ANI Supervisor ………….………………………… DR. P.O. UVERE Head of Department
6
DEDICATION
This work is dedicated to God Almighty for giving me the inspiration to persevere
And
To my beloved wife, Eucharia and son, Chiagozie who encouraged me to finish this study.
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ACKNOWLEDGEMENT
I am primarily grateful to God Almighty for His infinite mercies, provision and sustenance. My
greatest thanks go to my project Supervisor, Professor (Mrs.) J. C Ani for her constant advice,
assistance and technical involvement throughout the course of this work. I must also appreciate
her sweet husband for his fatherly care and concern. I am also indebted to the entire staff of the
Department of Food Science and Technology, University of Nigeria, Nsukka for their
assistance to me at the various stages of this research work. I am thankful to Mr. Oformata, the
Chief Technologist and his crew, particularly Mr. Hillary Asogwa and Mr. Ugwuanyi all of
Chemical Laboratory section of National Centre for Energy Research and Development,
University of Nigeria, Nsukka for allowing and assisting me to use their facilities.
I would like to express my deep appreciation to Prof. K. F. Chah, Head of the Department,
Mr. M. I.. Ngwu and Mrs. M. Onyishi, all in the Department of Veterinary Pathology and
Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, for their
invaluable material, technical and friendly assistance in the course of this work, particularly
during the antimicrobial screening. I am thankful to the entire staff, particularly the Head of
Department, Mrs. J.N. Ogara and the Chief Technologist, Mrs. Esther Ajayi. of the
Department of Basic Sciences, College of Agriculture, Lafia, Nasarawa State for allowing me
to use their facilities, particularly during the antimicrobial screening aspect of this work. I
appreciate Mr. E. Osuagwu and the entire Technical Staff of Crop Science Department,
University of Nigeria, Nsukka, and Mr. O.O Afolabi and his crew at the Biochemistry
Laboratory section of the Institute of Agricultural Research and Training (IAR&T), Ibadan,
Nigeria for their technical assistance and allowing me to use their Facilities for this study,
particularly during the chemical analysis.
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I also express my gratitude to my employer, Nasarawa State University, Keffi, for allowing me
time to do this work. I must appreciate my Head, Department of Home Science &
Management, Dr. H.J. Kaka and my Dean, Faculty of Agriculture, Prof. F.A. Ajayi for their
moral support and encouragement to persevere in the course of this work. Also to be heartily
acknowledged are my numerous relations, colleagues and friends, my beloved wife, Eucharia,
my son, Chiagozie and my daughter, Chidinma, my mother, Mrs. Agness Ugwuona and a host
of others, who have helped me financially and otherwise.
May God bless everyone who has contributed in any way towards the success of this study.
9
TABLE OF CONTENTS Title page ................................................................................................................... i
Approval page .......................................................................................................... ii
Certification .............................................................................................................. iii
Dedication ................................................................................................................. iv
Acknowledgement .................................................................................................... v
Table of contents ...................................................................................................... vii
List of figures ........................................................................................................... xii
List of tables ............................................................................................................. xiii
Abstract ..................................................................................................................... xv
CHAPTER ONE
1.1. Introduction ................................................................................................... 1
1.2. Statement of the Problem .............................................................................. 2
1.3. Justification for the Study .............................................................................. 3
1.4. Significance of the Study……………………………………. ...................... 3
1.5 Aims and Objectives ..................................................................................... 3
CHAPTER TWO
2.0 Literature Review ................................................................................................ 5
2.1. Spices………………………………………………………………………….. 5
2.2. Phytochemicals ……………………………………………………………….. 6
2.3. Classes of major phytochemicals, food sources and nutritional benefits ………… 7
2.4. Polyphenols ………………………………………………………………. … 9
2.5. Flavonoids ……………………………………………………………… 10
2.6 Anthocyanins ……………………………………………………………… 14
2.7 Carotenoids ……………………………………………………………….. 14
2.8 Ascorbic acid ………………………………………………………………. 16
2.9 Phytosterols ………………………………………………………………… 17
2.10 Phytoestrogens ………………………………………………………...….. 18
10
2.11 Phytochemical metabolism in human ………………………………………. 18
2.12 Lipid oxidation ……………………………………………………………. 19
2.13 Degenerative effects and suppression of lipid oxidation ………………. 21
2.14 Functions and mechanism of action of antioxidants in foods ………… 23
2.15 Natural antioxidants …………………………………………………….. 24
2.16 Antioxidant properties of spices and spice extracts ……………………... 25
2.17 Assessment of antioxidant activity of antioxidant compounds and the degree
of lipid Oxidation in food system………………………………………………... 26
2.18 Major microorganisms of food poisoning ………………………………. 28
2.19 Important preservation techniques for preventing food poisoning from
pathogenic microorganisms ………………………………………………. 28
2.20 Antimicrobial properties of spices and spice extracts …………………… 30
2.21 Choice of solvents for preparation of crude extracts from biological
Materials………………………………………………………………………. 31
2.22 Biology and ecology of Tetrapleura tetrapetra (Schum & Thonn) …………. .34
2.23 Nutrient composition of Tetrapleura tetrapetra …………………………………. 34
2.24 Food and medicinal uses of Tetrapleura tetrapetra ……………………………… 35
2.25 Ecology, botany and distribution of Monodora tennifolia (Benth) ……… … 36
2.26 Chemical composition and uses of Monodora tennifolia (Benth) …………… 36
2.27 Ecology, botany and distribution of African nutmeg (Monodora
myristica Gaetn) …………………………………………………………….. 37
2.28 Chemical composition and uses of African nutmeg
(Monodora myristica Gaertn) …………………………………………….. . 37
2.29 Ecology, botany and distribution of Ocimum viride (Willd) ……………... 38
2.30 Chemical composition and uses of Ocimum viride ……………………………. 38
CHAPTER THREE
3.0 MATERIALS AND METHODS…………………………………………… 40
3.1. Materials ........................................................................................................... 40
3.2 Preparation of spice extracts ………………………………………………….. 40
3.3 Preparation and storage of cooked ground beef and pork patties……………. 41
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3.4 Proximate analysis ……………………………………………………….. . . 42
3.4.1 Moisture content ………………………………………………………… … . 42
3.4.2 Crude protein content ............................................................................ 43
3.4.3 ether extract …………………………………………………….. ……….. 44
3.4.4 Crude fibre content ........................................................................... 44
3.4.5 Total ash content ................................................................................ 45
3.5 Energy value ........................................................................................ 45
3.6 Digestion and analysis for minerals ………………………………………….. 45
3.7 Determination of vitamin composition ………………………………………. 47
3.7.1 Determination of ascorbic acid (Vitamin C) content ……………………... 47
3.7.2 Determination of niacin content ………………………………………….... 47
3.7.3 Determination of riboflavin content ……………………………………….. 48
3.7.4 Determination of thiamin content ………………………………………….. 49
3.8 Determinations of phytochemical composition …………………………… 49
3.8.1 Determination of total phenol ……………………………………………… 49
3.8.2 Determination of total flavonoids …………………………………………… 50
3.8.3 Determination of condensed tannin content …………………………………. 50
3.8.4 Determination of total anthocyanin content ………………………………… 51
3.8.5 Determination of carotenoid content …………………………………………. 51
3.8.6 Determination of alkaloid content …………………………………………... 52
3.8.7 Determination of phytate content …………………………………………… 52
3.8.8 Determination of oxalate content ……………………………………………. 53
3.8.9 Saponin content determination ……………………………………………… 53
3.9 Determination of Antioxidant Properties of Spices ………………………….. 54
3.9.1 Determination of free radical scavenging activity ………………………….. 54
3.9.2 Measurement of reducing power of the crude extracts of spices ………… 54
3.9.3 Determination of antioxidant activity of crude extracts of the spices by the
Ferric thiocyanate (CTC) method ……………………………………….. 55
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3.10 Determination of antimicrobial properties of the spices ………………… 55
3.10.1 minimum inhibitory concentration (MIC) determination ……………….... 55
3.10.2 Preparation and inoculation of substrates ……………………………….. 56
3.10.3 Bacterial strains and preparation of inoculants…………………………... 57
3.10.4 Monitoring of survival and growth of pathogen population ……………. 57
3.11 Determination of Thiobarbituric acid (TBA) reactive substances in minced
meat patties during storage ……….…………………………………………… 58
3.12 Experimental design ………………………………………………………… 59
3.13 Statistical analysis …………………………………………………………… 59
CHAPTER FOUR 4.0 RESULTS AND DISCUSSIONS ……………………………………….. 60 4.1 Proximate composition and energy value of spices …………………………. 60 4.2 Mineral composition of the spices ……………………………………………. 64
4.3 Vitamin content of the spices ……………………………………………… 69
4.4 Yield of crude extracts of spices ……………………………………………….. 73
4.5 Effects of different extraction solvents on non-phenolic phytochemical content
of the spices …………………………………………………………………… 75
4.5.1 Alkaloid …………………………………………………………………… 75
4.5.2 Oxalate ……………………………………………………………………… 78
4.5.3 Saponin ……………………………………………………………………… 81
4.5.4 Phytate ……………………………………………………………………… 83
4.6 Effects of different extraction solvents on phenolic phytochemical contents
of the spices ……… ………………………………………………………………. 86
4.6.1 Total phenol content ………………………………………………………….. 86
4.6.2: Condensed tannin content …………………………………………………. 89
4.6.3 Total flavonoid content …………………………………………………….. 91
4.6.4 Total carotenoid content …………………………………………………… 94
4.6.5 Total anthocyanin content …………………………………………………… 96
4.7 Estimation of reducing power of spices …………………………………… 98
4.7.1 Reducing power of Ocimum viride ……………………………………………… 98
4.7.2 Reducing power of of Monodora myristica …………………………………… 101
4.7.3 Reducing power of Monodora trifolia …………………………………………… 103
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4.7.4 Reducing power of Tetrapleura tetrapetra …………………………………… 105
4.7.5 Comparative effect of extracting solvents on reducing power of the four spices 107
4.8 Inhibition of Linoleic acid peroxidation ………………………………… . 109
4.9.1 Scavenging of 1,1 – diphenyl-2-picryl hydrazyl radical (DPPH) by the spices 111
4.9.2 DPPH radical scavenging activity of Ocimum viride ………………………… 114
4.9.3 DPPH radical scavenging activity of Monodera myristica …………………… 116
4.9.4 DPPH radical scavenging activity of Monodora tenuifolia………………… 118
4.9.5 DPPH Radical scavenging activity of Tetrapleura tetrapetra ……………… 120
4.9.6 Comparison of DPPH radical scavenging activities of solvent extract of
spices……………………………………………………………………….. 122
4.10 Inhibition of lipid peroxidation in cooked, ground meat patties by spices
during storage…………………………………………………………………… 124
4.11 Comparison of Mean TBA values of meat patties treated with spices
during storage ……………………………………………………………… 126
4.11.1 Mean TBA values for Ocimim viride …………………………………….. 126
4.11.2 Mean TBA values for Monodora myristica ………………………………. 129
4.11.3 Mean TBA values for Monodora tenuifolia………………………………… 131
4.11.4 Mean TBA values for Tetrapleura tetropetra ……………………………… 134
4.12 Antimicrobial activities of the spices against some selected food pathogens…… 138
4.12.1 Sensitivity of the three bacteria toward inhibitory activity of aqueous
and ethanol extracts of the spices .......................................................... …. 138
4.12.2 Growth of pathogens in control substrates ………………………………… 141
4.12.3 Antibacterial activities of of Monodora myristica against food borne
Pathogens …………………………………………………………………….. 142
4.12.4 Antibacterial activities of Monodora tenuifolia against food borne
pathogens ….…………………………………………………………………… 148
4.12.5 Antibacterial activities of Ocimum viride against food borne pathogens ……... 154
4.12.6 Antibacterial activities of Tetrapleura tetrapetra against food borne pathogens ……
160
4.12.7 Phytochemical composition of cooked spice-treated food extracts…………….. 168
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS …………… …………………….. 173
14
5.1 CONCLUSION …………………………………………………………………… 173
RECOMMENDATIONS……………………………………………………………… 175
References ………………………………………………………………………….. 176
Appendices …………………………………………………………………………….. 193
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LIST OF FIGURES
1. Antioxidant activity of different solvent extracts of Ocimum viride……………… 100
2. Antioxidant activity of different solvent extracts of Monodora myristica ...……… 102
3. Antioxidant activity of different solvent extracts of Monodora trifolia ………... 104
4. Antioxidant activity of different solvent extracts of Tetrapleura tetrapetra ………106
5. Comparison of reducing properties of spices………………………………………108
6. Inhibition of linoleic acid per oxidation by different solvent extracts of spices….. 110
7. DPPH radical scavenging activities of different solvent extracts
(mg dry matter/mil) of Ocimum viride. ………………………………….. 115
8. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Monodora myristica……………………………………. .117
9. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Monodora trifoliaa.,…………………………………… 119
10. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Tetrapleura tetrapetra………………………………… 121
11. DPPH
radical scavenging activities of different solvent extracts
(mg dry matter/mil) of Ocimum viride (O. vir), Monodora myristica
(M.myr), Monodora tenuifolia (M. tri) and Tetrapleura tetrapetra (T. tetr). ….. 123
16
LIST OF TABLES
1. ...................................................................................................................... Phytochemic
al constituents of some plant foods and their functions ………..…. 8
2. ...................................................................................................................... Flavonoid
phytochemicals and their food sources …………………………. .… 12
3. ...................................................................................................................... Some
important flavonoid phytochemicals and their functions ……………… 13
4. ...................................................................................................................... Categorizatio
n of procedures to preserve foods from microbial spoilage ……….29
5. ...................................................................................................................... Some food
grade solvents and their physicochemical properties ……………… 29
6. ...................................................................................................................... Proximate
composition and energy value of spices ……………………….. 63
7. ...................................................................................................................... Mineral
composition (mg/100g) of spices …………………………………… 67
8. ...................................................................................................................... Vitamin
contents of spices ……………………………………………………… 72
9. Yield (%) of crude extracts of spices as affected by different extracting
solvents……………………………………………………………………. 74
10. Effect of different extraction solvents on alkaloid contents of spices…….…. 77
11. .................................................................................................................... Effect of
different extraction solvents on oxalate contents of spices…………. 80
12. .................................................................................................................... Effect of
different extraction solvents on saponin contents of spices ……… 82
13. .................................................................................................................... Effect of
different extraction solvents on phytate contents of spices ………… 85
14. .................................................................................................................... Effect of
different extraction solvents on phenol contents of spices………… 88
15. .................................................................................................................... Effect of
different extraction solvents on tannin contents of spices …....... 90
16. .................................................................................................................... Effect of
different extraction solvents on flavonoid contents of spices……. 93
17
17. .................................................................................................................... Effect of
different extraction solvents on carotenoid contents of spices……… 95
18. Effect of different extraction solvents on anthocyanin contents of spices…….. 97
19. .................................................................................................................... Free radical
1,1-diphenyl-3-picryl hydrazyl(DppH) Scavenging activity by
different solvent extracts of spices. …………………………………………… 113
20. .................................................................................................................... Inhibition of
lipid peroxidation in cooked, ground meat paties by spices…… 125
.
21. Interaction between cooked meat samples and O. viride spice levels on
Mean TBA values (milimalonaldehyde) during storage ……………………. 128
22. Interaction between cooked meat samples and M. myristica spice levels on
mean TBA values (milimalonaldehyde) during storage .................................. 130
23. Interaction between cooked meat samples and M. tenuifolia spice levels on
mean TBA values (milimalonaldehyde) during storage ………………… 133
24. Interaction between cooked meat samples and T. tetrapetra spice levels on
mean TBA values (milimalonaldehyde) during storage………………… . 137
25. .................................................................................................................... Minimum
inhibitory concentration (MIC) of aqueous and ethanol extracts
of Ocimum viride, monodera myristica, monodera trifolia and tetrapleura
tetrapetra against the test organisms. …………………………………………… 140
26. .................................................................................................................... Mean
microbial (Escherichia coli) population (106
x Cfu / ml) of aqueous
food extracts (10 %) treated with different levels of Monodora myristica
…………………………………………………………………………………. 143
27. .................................................................................................................... Mean
microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Monodora myristica. …… 145
28. .................................................................................................................... Mean
microbial (Staphylococcus aureus) population (106
x Cfu/ml) of
aqueous food extracts (10 % w/w) teated with different levels of Monodora
myristica……………………………………………………………………………... 147
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29. .................................................................................................................... Mean
microbial (Escherichia coli) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Monodora tenuifolia…………………
………………………………………………….. 149
30. .................................................................................................................... Mean
microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Monodora tenuifolia …….. 151
31. .................................................................................................................... Mean
microbial (Staphylococcus aureus) population (106 x Cfu/ml) of
aqueous food extracts (10 %) treated with different levels of
Monodora tenuifolia …. ………………………................................................. 153
32. .................................................................................................................... Mean
bacterial (Escherichia coli) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Ocimum viride………… 155
33. .................................................................................................................... Mean
microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Ocimum viride .. . ….. … 157
34. Mean microbial (Staphylococcus aureus) population (106 x Cfu/ml) of
aqueous food extracts (10 %) treated with different levels of Ocimum
vivide…………………………………………………………………………….. 159
35. .................................................................................................................... Mean
microbial (Escherichia coli) population (106 x Cfu/ml) of aqueous
food extracts (10 %) treated with different levels of Tetrapleura tetrapetra …….. 161
36. .................................................................................................................... Mean
microbial (Salmonella typhii) population (106 x Cfu/ ml) of aqueous
food extracts (10 %) treated with different levels of Tetrapleura tetrapetra ….. 163
37. .................................................................................................................... Mean
microbial (Staphyloccocus aureus) population (106 x Cfu / ml) of
aqueous food extracts (10 %) treated with different levels of Tetrapleura
tetrapetra…………………………………………………………………………. 166
38. Non-phenolic phytochemical profiles of water extracts of cooked
spice-treated foods………………………………………………………………………………… 170
39. Phenolic phytochemical profiles of water extracts of cooked
spice-treated foods………………………………………………………… 172
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ABSTACT This study evaluated phytochemical composition, antioxidant and antimicrobial properties of four Nigerian spices, namely Ocimum viride (leaves), Monodora myristica (seeds), Monodora tenuifolia (seeds) and Tetrepleura tetrapetra (fruits). The spices were screened for phytochemical [alkaloid, saponin, oxalate, phytate, total phenol (TP), condensed tannin (CT), total flavonoid (TF) and total anthocyanin (TA)] contents and antioxidant activities in five different extracting solvents [distilled water, 95 % methanol, acetone / hexane (1 : 1, v/v), hexane / methanol / acetone (2 : 1 : 1, v/v/v) and acetone / water / acetic acid (70 : 29.5 : 0.5, v/v/v)] using standard methods. Antioxidant capacities of the extracts to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, reduce iron (iii) chloride (FeCl3), suppress linoleic acid ( LA) peroxidation in ferric thiocyanate (FTC) oxidizing systems and inhibit formation of thiobarbituric acid reactive substances (TBARS) in refrigerated (4OC, 14 days) spice-treated (0, 0.6. 1.2 and 2.0%, w/w basis) beef and pork patties were investigated. Aqueous extracts (10% w/w) of beef, rice and vegetables were treated with the spices (0, 2.5 and 7.5%, w/v), inoculated with the pathogens Escherichia coli, Salmonella typhii and Staphylococcus aureus (5logCFU/ml), and periodically analysed, by enumerating surviving populations after 24h intervals, during 10 days of storage (4OC) to determine antimicrobial activities of the spices. Phytochemical contents differed significantly (p < 0.05) among the spices and among solvent extracts of the same spice. The highest phytochemical content was total Phenol which ranged from 2.13 garlic acid equivalent per100 g (GAE / 100 g) in M. myristica to 13.93 GAE / 100 g in T. tetrapetra while total anthocyanin content was the lowest and ranged from 0.00 GAE / 100g in M. tenuifolia to 0.06 GAE / 100g in M. myristica. The extracts of spices exhibited high degree of antioxidant and antimicrobial activities. The spices suppressed lipid peroxidation in cooked ground beef (from 2.58 to 0.76 mean thiobarbituric acid value) and pork patties (from 4.33 to 1.03 thiobarbituric acid value) in dose-dependent order during 14 days of storage. Spice extracts reduced Fe3+ to Fe2+, scavenged DPPH radical (78 – 93%) and inhibited LA peroxidation (46 – 95%) in dose-dependent order. Methanol (95 %) extracts of M. myristica, M. tenuifolia and O. viride, and water extract of T. tetrapetra exhibited the highest (1.6 nm) reducing power while the acetone/water/acetic acid extracts exhibited the highest (93%) scavenging capacity of DPPH radical. Water extracts of O. viride and T. tetrapetra, methanol extract of M. tenuifolia and acetone/water/acetic acid extract of M. myristica had the highest inhibition of LA peroxidation. The four spices exhibited dose-dependent bactericidal effects against E. coli (from 42.25 to 0.00 x 106 CFU / ml), S. typhii (from 47.1 to 3.7 x 106 CFU / ml) and S. aureus subsp.aureus (from 48.95 to 0.00 x 106 CFU / ml). During storage, antimicrobial effects of the spices were more pronounced in food extracts than in nutrient broth and in rice and vegetable extracts than in beef extracts. Of the four pathogens, E. coli was most susceptible to these spices, followed by S. aureus subsp. aureus. Tetrapleura tetrapetra was the most potent of these spices against the pathogens, followed by O. viride. The antioxidant and antimicrobial properties exhibited by these spices increased with spice concentrations and occurred in the following decreasing order: T. tetrapetra > O. viride > M. myristica > M. tenuifolia.
20
CHAPTER ONE
1.1 INTRODUCTION
In Nigeria, a high proportion of the rural and urban population resort to natural food
ingredients, particularly because of their availability. Spices are a large group of such natural
ingredients, and include dried seeds, fruits, roots, rhizomes, barks, leaves, flowers and any
other vegetative substances used in a very small quantity as food additives to colour, flavour or
preserve food (Birt, 2006). Spices are fragrant, aromatic and pleasant. The bulk of the spices
consist of carbohydrates such as cellulose, starch, pentosans and mucilage, and some amount
of protein and minerals (Ogutimein et al., 1989). Only very small fractions of dry matter of the
spices such as the phytochemicals are responsible for the flavouring, colourng, preservative
and health-promoting characteristics (Cowan, 1999).
These phytochemicals are plant metabolites (Sofowurra, 1993) which act as natural defense
systems for host plants, and also provide characteristic colour, aroma and flavour in specific
plant parts. They are a group of non-nutrient compounds that are biologically active when
consumed by human. Many phytochemicals are health-promoting and are of many disease-
preventive (Rowland, 1999; Birt, 2006). Both epidemiological and clinical studies have
proven that phytochemicals present in cereals, fruits and vegetables are mainly responsible for
reduced incidence of chronic and degenerative diseases among populations whose diets are
high in these foods (Shahidi, 1996). As a result there has been an increased search for
phytochemical constituents that possess antioxidant and antimicrobial potency in recent time
(Jayaprakasha and Jaganmohan, 2000, Birt, 2006). Typical phytochemicals with antioxidant
and antimicrobial activities include polyphenols, phenolic acids and their derivatives,
flavonoids, phospholipids, ascorbic acid, carotenoids and sterols. A number of exotic spices of
international recognition with known phytochemical constituents have been proven to be good
21
natural antioxidants (Dorko, 1994; Abd El-Alim et al., 1999; Seifried et al,, 2007),
antimicrobial (Mitscher et al., 1972; Billing and Sherman, 1998) and health-promoting agents
(Chan et al., 1995, Arai et al., 2000; Zhou et al. 2003). Some of such internationally
recognized spices include chili pepper, garlic, onion, anise, cinnamon, ginger, curry, rosemary
and nutmeg (Dorko, 1994; Arai et al., 2000; Birt, 2006).
However, there is paucity of information on the phytochemical compositions, antioxidant and
antimicrobial properties of many Nigerian spices which have been in use for centuries as
flavouring ingredients in many traditional dishes. Prominent Nigerian spices, including
Tetrapleura tetrapetra (Schum & Thonn), Monodora myristica (Gaertn), Monodora tenuifolia
(Benth) and Ocimum viride (Willd) need to be evaluated for these important properties for
broader application in food processing and preservation. The parts of these plants used as
spices are fruits of Tetrapleura tetrapetra, seeds of Monodora myristica and Monodora
tenuifolia, and leaves of Ocimum viride. The vernacular names of these spices in Igbo, Nigeria
are Ehuru for Monodora myristica, Ehu for Monodora tenuifolia, Hiohio for Tetrapleura
tetrapetra and Nchu-anwu or Ahunji for Ocimum viride.
The study is therefore designed to evaluate phytochemical compositions, antioxidant and
antimicrobial properties of solvent extracts of Tetrapleura tetrapetra (fruits), Monodora
myristica (seeds), Monodora tenuifolia (seeds) and Ocimum viride (leaves).
1.2 Statement of the problem
Information abound in literature on the phytochemical composition, antioxidant and
antimicrobial properties of many exotic spices and this promote their use internationally as
natural preservatives and as components of functional foods to promote health. Such
22
information is dearth on most indigenous Nigerian spices and this limits their use
internationally as preservatives and functional ingredients. It is therefore necessary to evaluate
phytochemical compositions, antioxidant and antimicrobial properties of some popular
Nigerian spices, namely Tetrapleura tetrapetra, Monodora myristica (Ehuru), Monodora
tenuifolia (Ehu) and Ocimum viride (Nchu-anwu) to diversify their use as natural preservatives
and as culinary spices that contain active ingredients that promote health and reduce the risk of
disease.
1.3 Justification for the study
The paucity of knowledge of the phytochemical constituents, antioxidant and antimicrobial
properties of these indigenous herbs and spices has resulted in their neglet and underutilization.
It is envisaged that the result of this study will initiate the exploitation of the preservative,
nutraceutical and therapeutic potentials of these culinary herbs and spices.
1.4 Significance of the study
This study will provede detailed information on phytochemical compositions, antioxidantand
antimicrobial of these four spices for broader application in foods and other relevant areas. The
spices, with information on phytochemical, antioxidant and antimicrobial properties, would
attract international recorgnitions that can earn Nigeria huge revenue. This would create
employment for many Nigerians who would propagate and process the spices.
1.5 Aims and objectives
The broad objective of this study was to evaluate the potentials of four local Nigerian spices
for food preservation and promotion of good health.
23
The specific objectives of the study were to:
1. Evaluate phytochemical composition of Tetrapleura tetrapetra, Monodora
myristica, Monodora tenuifolia and Ocimum viride;
2. Evaluate antimicrobial properties of the four spices against strains of Escherichia coli,
Salmonella typhii and Staphylococcuss aureus in nutrient broth, vegetable mix, beef
and parboiled rice extracts;
3. Evaluate antioxidant properties of the spices in scavenging and reducing activities, and
in suppressing peroxidation in linoleic acid and in cooked meat samples.
24
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1. Spices
Spices are indispensable components of cuisines used mainly for flavouring to improve
palatability of food (OKigbo, 1977; Okafor, 1987). A spice is a dried seed, fruit, root, bark,
flower, leave or any vegetative substance used in a very small quantity as food additive to
colour, flavour or preserve food. The United States Food and Drug Administration (FDA)
defined spices as aromatic vegetative substances used for seasoning of food and from which no
portion of any volatile oil or flavouring principles have been removed, and are free from
artificial colouring matters, adulterants and impurities (Farrel, 1990). Spices are ‘‘Generally
Recognized As Safe’’ (GRAS) by the FDA, at least at concentrations commonly found in
foods.
Spices contribute very minimal nutrients to menu because they are used a very small amount.
The bulk of the major components of spice materials consist of carbohydrate, protein and little
minerals. Tannins, resins, pigments, volatile, essential and fixed oils which contribute to
flavouring occur in traces and constitute only a small fraction of the dry matter (Cowan, 1999).
Some well known spices of commerce include red pepper, onions, sage, ginger, nutmeg, clove,
cinnamon, mustard, curry, turmeric, rosemary and garlic. Spices add flavour, relish and
pungency to diets. Most spices are fragrant, aromatic and pleasant. Spices in food also exert
such secondary effects as salt and sugar reduction, prevention of spoilage and improvement of
texture (Ravindran et al., 2002).
25
Spices, because of many health-promoting phytochemicals they contain are known to fight
cancer and many heart diseases. Apart from being used as food ingredients, spices are also
used as components of many medicines, perfumery, cosmetics and natural colours. Many
spices have been in use in many traditional medicines. Many ethnic cuisines are highly
recognized for their reliance due to some perculiar spices in them. The turmeric in Italian
cuisine, basil, garlic, and oregano in Italian and Greek cuisine, and lemon grass, ginger and
chili peppers in Thai foods are examples of spices used in different cultures (Satia-Abouta et
al., 2002).
2.2 Phytochemicals
Phytochemicals consist of a large group of naturally occurring non nutrient, biologically active
compouds found in plants. As implied by the prefix “phyto” in the name, phytochemicals are
basically produced only by plants. Phytochemicals act as natural defense system for the host
plants and in addition provide colour, aroma and flavour. Plants use phytochemicals as natural
protection from bacteria, fungi and viruses (Ramanthan et al., 1989; Duyff, 2000). More than
4000 of these compounds have been discovered and it is expected that scientists will yet
discover many more phytochemicals in plant foods such as fruits, vegetables, legumes, cereals,
herbs and spices (Rowland, 1999; American Institute of Cancer Research, 2000).
Phytochemicals give hot pepper the burning sensation, onions and garlic the pungent flavour
and tomatoes their red colour. Phytochemicals can have profound physiological effects, act as
antioxidants, mimic body hormones and suppress development of diseases in the body (Milner,
2002; Lesschaeve and Noble, 2005; Hayes, 2005). Phytochemials are needed in daily meals
for proper healthy living.
26
2.3. Classes of major phytochemicals, food sources and nutritional benefits
Phytochemicals are numerous and are found in all plant products, including fruits, vegetables,
legumes, cereals, herbs and spices (Rowland, 1999; American Institute of Cancer Research,
2000). No single plant material is naturally endowed with all the vital phytochemials needed
by human. Consequently, it is advised that a wide variety of plant materials, including fruits,
vegetables, grains, herbs and spices be consumed in order to benefit maximally from the rich
combination of phytochemicals (American Institute of Cancer Research, 2000).
Phytochemicals are preferably sourced from a variety of plant materials rather than from food
supplements or pills (Rowland, 1999). Food supplements and pills provide only very few of
the thousands of phytochemicals needed, and thus are less effective than a single serving of
fruits and vegetables (Milner, 2002). Most of the plant materials in human diets contain some
important phytochemicals. Some good food sources of phytochemicals include cabbage,
lettuce, tomatoes, Carrot, water melon, mangoes, pawpaw, grapes, oranges, apples, cashew
apple and nut, mustard, pears, oats, sweet potatoes, whole wheat, beans, ginger, onions, red
pepper spinach, sesame seed and garlic among others (Bouguet and Debray, 1974; Herber and
Lu, 2002; Hayes, 2005). According to Birt (2006), phytochemicals work in synergy and their
effects when served together are stronger than the sum of the effects of parts served separately.
The thousands of phytochemicals so far discovered are group-based. Table 1 shows some of
these phytochemicals, their sources and biological functions.
27
Table 1: Photochemical constituents of some plant foods and their functions.
Phytochemical classes
Phytochemicals Sources Potential nutritional benefits
Carotenoids β-carotene,& carotene, lutein, lycopene
Tomato, pumpkin, carrot, water melon. Guava, dark yellow pink and red coloured vegetative fruits
- Act as antioxidant - Reduce level of cancer - Producing enzymes - Inhibit spread of cancer
Saponins Panaxadiol, panaxatriol
Potato, tomato, soybean, beans
Reduce glucose and glycerol uptake in the gut.
Flavonoids Anthocyanin, anthoxanthins
Beans, citurs fruits - Block access of carcinogen, prevents
Malignant change in cells. Prevent cancer
Polyphenols Tannin Fruits, vegetables, legumes
Exhibit anti-microbial and anti-oxidant activities
Green vegetable black tea
- Increase antioxidant activity. Prevent proliferation of cancer
- help speed excretion of carcinogen from the body
Phytic acid Fruits, potatoes, soybeans, whole grains and legumes
Bind iron and prevents it from forming cell damaging free radicals
Flavonoid Anthocyanins Fruits and green vegetables
Act as anti-oxidants, improve balance in co-ordination and short time memory, and prevent urinary tract infection.
Phytosterols β-sitosterol, campesterol, stigmasterol
Potatoes, tomatoes, vegetable oils, alfafa sprout
Block excess uptake of dietary cholesterol and facilitate cholesterol excretion
Terpenes Mono-terpenes Garlic, maize, ginger Help detoxify carcinogens, inhibit spread of cancer.
Carotenoids Limonenes, carvones Β-carotene, Lutein, Lycopene, Zeaxanthin
Citrus fruits, water melon, carrot, mango,
Help detoxify carcinogens, inhibit spread of cancer cells.
Isothiocyanates Allylisothiocyanate, indoles,sulfuraphane
Cruciferous vegetables including cabbage, curli flower, broccoli
Suppress tumour growth, boost proliferation of cancer-fighting enzymes.
Organosulphides Diallyl Sulphide, AllymethylSulphide, S-allylcysteine.
Garlic, onions, braccoli, cabbage, mustard.
May slow production of carcinogen but speed production of carcinogen-destroying enzymes
Carrotenoids Lycopenes Tomatoes, watermelon, guava
Anticarcinogenic, inhibit proliferation of cancer cells.
Carrotinoids Β-carrotene Tomatoe, pumpkin, carrot, guava, pawpaw
Act as anti-oxidant, reduce risk of heart and eye-related diseases.
Rowland, (1999); Duyff, (2000); Herber and Lu, (2002); Birt, (2006)
28
2.4. Polyphenols
Polyphenols, which include more than 8,000 compounds, are a family of natural compounds
widely distributed in the outer layers of plant as suspected from their protective function in the
plants (Manach et al., 2004). Polyphenols occur in all plant foods and contribute to the
beneficial health effects of vegetables and fruit. They range from simple molecules such as
phenolic acid to highly polymerized compounds, such as tannins. Phenolic acids account for
about one third of the total intake of polyphenols in human diet. These compounds are capable
of removing free radicals, chelating metal catalysts; activate antioxidant enzymes, reducing α-
tocopherol radicals, and inhibiting oxidases (Oboh, 2006). As a result, they neutralize free
radicals formed during normal physiological functioning of human body (Burns et al., 2001;
Benzie, 2003). The antioxidant activity of phenols is due to their redox properties through
which they act as hydrogen donors, singlet oxygen quenchers, reducing and metal chelating
agents. There is a highly positive relationship between total phenols and antioxidant activity of
many plant materials (Gulcin et al., 2004). Daily consumption of polyphenols in the US ranges
from 200 mg to 1 gram. Green tea polyphenols and polyphenols from wine have attracted
media attention.
The antioxidant capacity of polyphenols in any diet is much higher than the combined
antioxidant effect of beta-carotene, vitamins A and E in the same diet (Gulcin et al., 2004). The
total intake of polyphenols in a person’s diet could amount to 1g per day, whereas combined
intakes of beta-carotene, vitamins C, and vitamin E from food most often is about 100 mg per
day (King and Young, 1999; Gulcin et al., 2004; Shahidi and Naczk, 2004). Important dietary
sources of polyphenols include onions (flavonols), Cocoa (proanthocyanidins), tea, apples and
red wine (flavonols and catechins), citrus fruit (flavanones), berries and cherries
(anthocyanidins), and soybean (isoflavones) (Oboh, 2006; Zahin et al., 2009). Polyphenol such
29
as gallic acid and cathechin from natural products are used as standards when determining total
phenol contents of plants and plant materials (Zahin et al., 2009).
2.5. Flavonoids
The polyphenols can be divided into a variety of classes, depending on the classification
system. One of the largest and very important of these classes is the flavonoids, which is made
up of several subclasses, namely flavonols, flavanols, flavones, flavonones, anthocyanidins and
isoflavones (Rowland, 1999; King and Young, 1999; Manach et al., 2004).
Flavonoids are found in cell membranes, between aqueous and lipid bilayers. Being water-
soluble, they are found in the cell sap. Flavonoids are typically categorized based on their
chemical structures. Monomer forms are called catechins while condensed forms are called
proanthocyanins (and also called tannins) (Briskin, 2000; Yeum and Russel, 2002). Tannins
provide astringency properties to foods and beverages. Flavonoids commonly found in fruits
and vegetables include quercetin, Luteolin, Kaempferol, hesperetin and cyanidin (Leighton et
al., 1992). Most fruits and vegetable do not contain luteolin and quarcetin together (Hurdson
and Lewis, 1983; Hertog et al., 1992).
Flavonoids are found in almost all plant based foods and beverages, but the levels vary,
depending on the degree of ripeness of the fruits, variety and processing. Most flavonoids
enhance the potency of vitamin C (ascorbic acid) and function as antioxidants. Antioxidant
activity of flavonoids is believed to be due to their ability to act as free radical acceptor and to
complex metal ions (Hertog et al., 1992). They are biologically active against liver toxins,
tumours, viruses and other microbes, allergies and inflammation (Ramanthan et al., 1989;
Cowan, 1999; De et al., 1999). Flavonoids protect the body blood cells, especially the tiny
30
capillaries that carry oxygen and nutrients to various parts of the body. Flavonoids are believed
to slow down the development of cataracts in diabetics. Table 2 shows some flavonoid
subclasses, specific examples of such subclass and their food sources while Table 3 shows
specific flavonoid phytochemicals and their functions.
31
Table 2: Flavonoids and their food sources
Flavonoids subclasses Food sources
1 Flavonones i. Hesperetin - orange
ii. Eriodicatol - Lemon
iii. Neringenin - Grapefruit
2 Flavones i. Lutelin - Parsley, some cereals
ii. Epigenin - Some cereals
3 Flavonols i. Quercetin - Onion, tea
ii. Kaempfeerol - Red wine, apple.
4 Flavanols i. Catechin (monoma) - Green tea
(also called anthocyanins), 3–
hydroxy derivatives
ii. Proanthocyanin (also
called tannin)
5 Anthocyanidin (unconjugated
aglycone anthocyanin)
Cyanidin Berries, red wine, cherries
6 Isoflavone Genistein,
Daidzein
Equol
Legumes
Soybean nuts,
soy sauce.
Sources: Pratt and Watts, (1963); Manach et al., (2004); Gu et al., (2004); Rowland, (1999); King and Young, (1999).
32
Table 3: Some important flavonoids and their functions
S/No Flavonoid Function
1 Hesperitin -Raises blood level of the “good cholesterol and lowers blood
level of the “bad” cholesterol
- Prevents inflammation and relieves pains
2 Quercetin Can prevent incidence of head and neck cancers
Protects the lungs from harmful effects of pollutants and
cigarette smoke
Reduces inflammation associated with allergies
3 Tangeritin - Induces cell death in cancer cells (Leukemia) but promotes the
life of normal healthy cells
4 Resveratol - May reduce the risks of heart diseases, stroke and blood clots.
5 Flavanols
(Anthocyanins)
Act as potent antioxidant
Helps to improve balanced coordination and short term
memory in the elderly
6 Anthocyanins Helps to prevent urinary tract infection.
Source: Hurdson and Lewis, (1983); Hertog et al., (1992)a.
33
2.6 Anthocyanins
Anthocyanins are water-soluble natural pigments (red, blue or purple) of plants, responsible
for the attractive colours of flowers, fruits (particularly berriers) and vegetables. They
contribute largely to the aesthetic quality of plant-derived products. A 100 g serving of berries
can provide up to 500 mg of anthocyanins in the diets (Manach et al., 2004). The anthocynins
are glycosides of polyhydroxy and polymethoxy derivatives of 2– phenylbenzopyrylium or
flavilium salts. Glycosylation and acylation of the aglycone moieties of the six main
anthocyanidins (pelargonidin, cynanidin, peonidin, delphinidin, petunidin, and malvidin) by
different sugars and acids at different positions, account for the broad structural diversity of
these pigments. Plant’s anthocyanins may enhance resistance to insect attack (Smirnoff and
Wheeler, 2001). According to Fuleki and Francis (1968), it has been recognized that
anthocyanin-rich plant extracts might serve as potential natural food colorants, especially when
tactically purified and made stable. Anthocyanin is high in plants with bright colours such as
berries (Fuleki and Francis, 1968).
2.7 Carotenoids
Carotenoids are diverse groups of lipophilic polyene compounds that are made up of 3 to 13
conjugated double bonds, and in many cases have a 6-carbon ring structure attached at one or
both ends of the molecules. They are a large family of yellow, orange and red pigments, all
soluble in fat and are found along with chlorophyll in green leaves, and in carrot, tomato,
peppers, banana and peaches. Carotenoids that contain oxygen in their chemical structures are
known as xanthophylls while the non-oxygenated ones are called carotenes (McClement et al.,
2007). Common examples of xanthophylls are lutein and zeaxanthin while common examples
of carotenes are lycopene and β-carotene. β –carotene, a brilliant orange-yellow pigment, is a
34
provitamin A. It is closely related to vitamin A and easily isomerizes to vitamin A when
consumed.
Carotenoids generally are widely distributed in the plant kingdom. They are found in most
green leaves, yellow and red coloured fruits, and in many roots and tubers (Gu et al., 2004;
Chee et al., 2005). They are not synthesized by animals but when ingested may be absorbed
undamaged or transformed to ’animal’ carotenoids. For example, the colour of egg yolk, some
plumage feathers, crustaceans, goldfish and salmon are due to the presence of different
carotenoids. White potato, butter, carrot root, egg yolk and sweet potato are common foods
with high contents of β-carotene. Lycopene is the red pigment in tomato, water melon and red
pepper, and has a slight vitamin A activity when eaten in diets (Stringham and Hammond,
2005). Lutein and zeaxanthin are thought to decrease age-related molecular degradation and
cataracts while lycopene is thought to decrease the risk of prostate cancer (Basu and Iurhan,
2007).
Carotenoids are highly unsaturated and are therefore susceptible to oxidation. Endogenous
carotenoids in food are generally stable but as food additives, carotenoids become relatively
unstable in the same food systems (Ribeiro et al., 2005). They are susceptible to light, oxygen
and heat (Yeum and Russel, 2002; Ribeiro et al., 2005; 2006). The conjugated double bonds in
carotenoid molecules can undergo isomerisation to the cis configuration. Isomerisation
reaction may be beneficial since the cis isomers of carotenoids such as lycopene are thought to
be more bioavaiable and bioactive than the trans isomers (Schieber and Carle, 2005).
Green vegetables must be blanched prior to freezing if off-colour, which oxidized carotenoid
breakdown products contribute, must be avoided. Canned commodities will retain their
35
carotenoid content without serious loss. In frozen foods, carotenoid retention is high unless an
unsaturated fatty acid oxidase, capable of destroying carotenoid pigments is present. Loss of
colour in carotenoid is possible due to the isomerisation of all trans forms to cis forms; and this
is promoted by light, heat and acid. The carotenoid content of food samples will be best
retained if stored at low temperature, inert atmosphere, blanched prior to storage, or stored
with added antioxidants (Ribeiro et al., 2005).
2.8 Ascorbic acid
Ascorbic acid (C6H8O6), commonly called vitamin C is a monosaccharide redox catalyst found
in both animals and plants. As one of the enzymes needed to synthesize ascorbic acid in human
has been lost by mutation during primate evolution, humans must obtain it from the diets as
vitamin (Smirnoff, 2000). Most other animals are able to synthesize this vitamin and do not
require its supply through diets (Linster and Van Schanftingen, 2007).
Ascorbic acid is required for the conversion of procollagen to collagen by oxidizing proline
residues to hydroxyl proline. In other cells, it is maintained in its reduced form by reacting with
glutathione, which can be catalysed by protein disulfide isomerase and glutathione (Meister,
1994; Well et al., 1990). Ascorbic acid is a redox catalyst which can reduce, and thereby
neutralize reactive oxygen species such as hydrogen peroxide (Padayatty et al., 2003). In
addition to its direct antioxidant effects, ascorbic acid is also a substrate for the redox enzyme,
ascorbate peroxidase which is particularly important in stress resistance in plants (Shigeoka et
al., 2002). Ascorbic acid is present in high levels in all parts of plants and can reach
concentrations of 20 mg in chloroplasts.
36
Ascorbic acid is added during the manufacture of juices or soft drinks to improve their
nutritional value or to prevent the autoxidation of such commercial products. It is a food
substance needed by humans to prevent scurvy, a disease of the bones, and blood vessels, and
to increase the body’s resistance to infection. Ascorbic acid occurs naturally in many fruits and
vegetable, particularly in tomatoes, mango, spinach, citrus fruits, green peppers, water melon,
cabbage, potatoes and broccoli. As an antioxidant, it prevents cataracts and cancer of stomach,
mouth, throat, and pancreas. It may also prevent the oxidation of low density lipoprotein
(LDL) cholesterol, lowering the risk of heart diseases (Nes and Skejelkvale, 1982; Nakatani,
2003).
2.9 Phytosterols
Phytosterols are a group of phytochemicals that include compounds such as stigmasterol, (3-
sitosterol and campesterol). Phytosterols occur in most plant species but are most abundant in
the seeds of green and yellow vegetables. Phytosterol concentration in vegetable oils range
from 0.1 % to 1.0 % (Chaiyasit et al., 2007) and typical phytosterol consumption is in the
range of 200 to 400mg/day. The production of phytosterol-fortified foods has become popular
due to the ability of phytosterols to decrease total and low-density lipoprotein (LDL)
cholesterol in humans by inhibiting the absorption of dietary cholesterol (Ostlund, 2004).
Dietary intake of 1.6 g phytosterol results in reduction of approximately 10% LDL cholesterol
(Hallikainen et al., 2000). Phytosterols modulate the level of dietary cholesterol absorbed by
the body by blocking uptake in the small intestine. They also facilitate cholesterol excretion
from the body, thereby reducing the risk of heart diseases. Absorption of phytosterols in the
small intestine is very low, so dietary intake of phytosterols hardly results to adverse effects on
human health.
37
Phytosterols are difficult to incorporate into foods because they have high melting point and
tend to form crystals. They are better esterified to polyunsaturated fatty acids which are more
soluble. Interestingly when these phytosterol esters are ingested, lipases hydrolyse them to
produce free phytosterols. Phytosterol oxidation products have been observed in model food
systems, oils and food products (Dutta, 1997). However, it is not clear whether oxidized
phytosterols lose their bioactivity or are toxic in a manner similar to oxidized cholesterols.
Dutta (1997) noted that encapsulation of phytosterol could increase its oxidative stability.
2.10 Phytoestrogens
Phytoestrogens are naturally occurring plant compounds that structurally resemble mammalian
estrogen in the body. Isoflavones are very common phytoestrogens abundant in soybean and
kudzu plants, and as popular as dietary phytoestrogen. Regular intake of dietary isoflavone
reduces risk of cancer, improves cardiovascular and bone health. Soy isoflavones are believed
to be useful in treatment of menopausal symptoms in women.
2.11 Phytochemical metabolism in human
Most phytochemicals found in foods exist in a variety of forms which influence their digestion
and absorption. Most common ones are the polyphenols which exist as glycoside conjugates.
Some glycosides must be digested to aglycones (unconjugated forms) before being absorbed
(King and Young, 1999; Manach et al., 2004). Some other forms of phytochemicals are
thought to be absorbed in the intestines without intensive digestion. The absorption of most
phytochemicals is thought to involve a carrier. Also, many glycosides are neither digested nor
absorbed in the small intestines. Such phytochemicals not absorbed in the small intestine have
38
been shown to undergo microbial degradation by colonic microflora (Rowland, 1999; Ross and
Kasum, 2002). The bacteria hydrolyse the glycosides, generating aglycones which may
undergo further metabolism to form various aromatic compounds (Bradlaw et al., 1999; Visioli
et al., 2000).
Once absorbed, most phytochemical metabolites get conjugated in the small intestine or in the
liver (Rhodes, 1996; Rowland, 1999). Conjugation most often involves methylation, sulfating
or glucunnidation. These conjugated metabolites are then bound to plasma proteins such as
albumin and are transported through the blood to various parts of the body (King and Young,
1999). The amount of these conjugated metabolites in the plasma varies considerably with the
type of polyphenol consumed, the food source, and the amount ingested (Briskin, 2000).
However, after consumption of specific polyphenols, little is known about the metabolism of
the different polyphenols in the body, and also about what metabolites are present in the
plasma.
2.12 Lipid oxidation
Oxidation of lipids which occur during storage, processing and heat treatment is one of the
basic processes causing rancidity in food products leading to oxidative deterioration (Hurdson,
1990). Oxidative deterioration of foods manifest in losses in colour, flavour, texture and
nutritive values of the food. The consequences of lipid oxidation include decreased nutritional
and physiological value of lipids and deterioration of fat-soluble vitamins and essential fatty
acids (Karpinska et al., 2001). Products of lipid oxidation have also been shown to cause
pathological changes in the mucus membrane of the alimentary tract, inhibit the activity of
enzymes and increase the contents of cholesterol and peroxides in blood system, thus
activating the process of atherosclerosis (Gardner, 1979; Karpinska et al., 2001). Lipid
39
oxidation products can also have carcinogenic activity (Gardner, 1979 Ames, 1983, Jacob,
1995).
Oxidative stability of food is related to the degree of saturation of the lipid fraction and the rate
of oxidation increases with the degree of unsaturation. Unless mediated by other oxidants or
enzymes, oxidation precedes through a free radical chain reaction mechanism involving three
stages namely:
1. Initiation, resulting in formation of free radicals. An unsaturated hydrocarbon loses
hydrogen atom to form a free radical required to start the propagation reaction, and
oxygen adds to the double bond to form a diradical as follows:
i. RH R* + H*
ii.
2. Propagation, results in free radical chain reaction to form peroxy radical (RO)
hydroperoxide (ROOH) and new hydrocarbon radicals (R.)
R + O2 ROO*
ROO* + RH ROOH + R*
The new free radical formed contributes to chain reaction by reacting with
another oxygen molecule (O2).
3. Termination, results in formation of stable, non-radical products by the inter-
reaction of the free radicals.
R* + R* RR
ROO* + ROO* - ROOR + O2
RO* + R* ROR
ROO* + R* ROOR
2RO* + 2ROO* 2 ROOR + O2
O - O
H H H H
R1-C = C-R2 + O2 R1 – C – C – R2
40
Antioxidants can be added to the food system to suppress the rancidity development. Oxidation
of lipids is accelerated by:
� Generation of heat and light energy,
� Presence of divalent metal catalyst,
� Presence of inherent enzymes in the system,
� Oxygen concentration and type of oxygen.
2.13 Degenerative effects and suppression of lipid oxidation.
Oxidation of lipid involves the peroxidation of polyunsaturated fatty acids located in the
membranes of living cells or in food systems (Keller and Kinseller, 1973; Adegoke et al.,
1998). In food system, oxidation of lipids results in decreased colour, flavour and texture,
nutritive and physiological values of lipids and lipid-soluble nutrients (Hurdson, 1990). Fat-
soluble vitamins and essential fatty acids are destroyed during lipid oxidation (Karpinska et al.,
2001). Oxidized foods become rancid and unacceptable. Oxidants from oxidized foods also
cause pathological changes in the mucus membranes of alimentary tracts, inhibit enzyme
activities, and increase cholesterol and peroxide contents in foods (Gardner, 1979; Ames,
1983; Jacob, 1995; Karpinska et al., 2001)
In living cells oxidation leads to production of energy necessary for essential cell activities.
Oxygen derived free radicals, commonly known as reactive oxygen species (ROS) are also
produced (Adegoke et al., 1998). ROS are by-products of normal body metabolism but if not
controlled may cause oxidative stress on the organs. Oxidative stress is associated with the
41
development of many chronic and degenerative diseases, including cancer, arteriosclerosis,
neuronal degradation, hypertension and aging (Ames et al., 1995; Christen, 2000).
Antioxidants are substances which delay or inhibit oxidative damages. Antioxidants either
inhibit the formation of free alkyl radicals at the initiation step or interrupt the propagation of
free radical chain reaction. Most commercial antioxidants in use are monohydroxy or
polyhydroxy phenolic compounds with various ring substitutions (Decker et al., 1992). They
require low activation energy to be able to donate hydrogen or antioxidant free radical ring to
the lipid free radical to form stable compounds. Commercial antioxidants in use in food
systems are of two categories, synthetic and natural antioxidants. Generally Recognized as
Safe (GRAS) synthetic antioxidants in use include butylated hydroxylanisol (BHA), butylated
hydroxyl toluene (BHT), pyrogallic acid (PG) and tertiary butyl hydroxyl quinone (TBHQ).
The natural antioxidants in commercial use include tocopherols (delta > gamma > beta >
alpha), nordihydrogurtic acid (NDGA), sesamol and gossypols.
Natural antioxidants are perceived by consumers to be better and safer than synthetic ones
(Dorko, 1994). Common natural antioxidants in use include tocopherol (delta > gamma > beta
> alpha), beta-carotene, ascorbic acid, sesamol, nordihydrogurtic acid and gossypol. Some
conventional spices of International Trade that exhibit antioxidant properties include rosemary
(Rosmarinus officinalis), sage (Salvia officinalis), garden thyme (Thymus vulgaris), oregano
(Origanum vulgare) and majoram (Origanum majoram), and so many others (Madsen and
Bertelsen, 1995; Ramarathnam et al., 1995). Ascorbic acid, tocopherol isomers, Carotene and
rosemary extracts have been used extensively in foods for their antioxidant properties
(Chipault et al., 1956, Lee, 1995; Lee and Shibamolo, 2001). Tocopherols, ascorbic acid and β-
42
carotene have been reported as compounds that may protect consumers against cancer, heart
disease and cataracts (Barrak and Langseth, 1994).
2.14 Functions and mechanism of action of antioxidants in foods.
Antioxidants are chemicals which delay the start or slow the rate of lipid oxidation reaction in
food systems. They need low activation energy to be able to donate hydrogen to free radicals to
form stable complexes. When the hydrogen is donated, the resulting antioxidant free radical
ring cannot initiate another free radical formation because they are stabilized. There is
delocalization of radical electron in the antioxidant free radical ring. The antioxidant free
radical ring cannot also undergo rapid oxidation but can react with lipid free radicals to form
stable complex compounds. Thus, antioxidants inhibit oxidative reaction of lipids in food
system by either donating hydrogen to free radicals to form stable compounds or by formation
of a complex between the antioxidant radical (ring) and lipid radicals viz.
1. R* + AH RH + A*
RO* + AH ROH + A*
2. ROO* + AH ROOH + A*
R* + A* RA
RO* + A* ROA,
Where R*, RO*, ROO* are respectively alkyl, alkoxy and peroxy radicals from lipid molecules, AH and A* are antioxidant and antioxidant radical respectively.
Application of antioxidants to foods really depends on the nature and composition of the food
system and also on the antioxidant and its composition. Antioxidants are applied to foods by
either mixing the antioxidant with oil or melted fat which is then mixed with the food, adding
the antioxidant in diluent which is then added to the food system, making a solution of the
43
antioxidant and then spraying to the food system or dipping food into the system. An ideal
antioxidant, whether natural or synthetic, should have the following features:
� It must have no harmful physiological effect on the consumers;
� It must contribute no objectionable flavour, odour, colour or taste to the food;
� It must be fat soluble and effective at low concentration;
� It must not be affected or destroyed by processing operations.
Other agents including metal chelators, singlet oxygen inhibitors and peroxide stabilizers also
inhibit lipid oxidation. Metal chelators in use include phosphoric acid, citric acid, ascorbic acid
and their salts and ethylene diamine – tetra – acetate (EDTA). They work by deactivating free
trace metals or metals of salt of fatty acids by forming complex ions or coordinated
compounds. Products from maillard reaction and smoking of meat and fish constitute some of
the singlet oxygen inhibitors and peroxide stabilizers.
2.15 Natural antioxidants
Antioxidants can be of natural or synthetic origin. Some synthetic antioxidants, such as BHA
and BHT, might be dangerous for living organisms (Attmann et al., 1986). Also naturally
derived antioxidants from plant materials, including cereals, legumes, fruits, vegetables, herbs
and spices are perceived by consumers to be better and safer than the synthetic antioxidants
(Dorko, 1994). Natural antioxidants are seasonal in supply but are mostly cheaper and easier to
use than the synthetic ones. They could be made readily available all year round if properly
processed and preserved during harvest.
44
2.16 Antioxidant properties of spices and spice extracts
Many spices such as cinnamon, clove, onion, turmeric and black pepper commonly used in
traditional cuisines exhibit antioxidant properties in different biological systems. Spices with
antioxidant properties have been shown to posses substances such as phenols and phenolic
acids, flavonoids, terpenes, terpenoids and lignans (Craig, 1999). The prominent antioxidant
compounds of specific spices include cinnamic aldehyde in cinnamon which gives it distinct
flavour and aroma (Murcia et al., 2004); capsaicin (Trans-8-methyl-N-vanillyl-6-none
enamide) and phenolic acids in chili pepper (Jimenez et al., 2000); and piperine, chavicine,
isopiperine, isochavicine and monoterpenes in black pepper (Milbourne, 1987). The
antioxidant property of chili pepper is attributed partly to the presence of ascorbic acid, many
flavonoids and phenolic acids (Jimenez et al., 2000). Cinnamon was shown to be a better
superoxide radical scavenger than mint, anise, butylated hydroxyanisol (BHA) and butylated
hydroxytoluene (BHT) (Murcia et al., 2004). The antioxidant compound in coriander
(Coriandrum sativum) is linalool (Reddy and Lokesh, 1992) while those in cloves (Syzygium
aromaticum) are eugenol and eugenylacetate (Lee and Shibamolo, 2001).
Fennel (Feniculum vulgane) has 3 – caffeoylquinic acid, vosmiric acid and quercetin – 3 – 0 –
galactoside as antioxidant compounds (Parejo et al., 2004) while ginger has gingerol-related
compounds and diarylheptanoid as the antioxidant compounds (Parejo et al., 2004). Nutmeg
(Myristica fragrance Houtt) has about 10% essential oil which is primarily composed of
terpene hydrocarbons (Pinenes, camphene, P-cymene, subinene, phellandrene, terpene,
limonene and myrcene; 60 – 90 %), terpene derivatives (Linalool, geranoil, and terpineol, 5 –
15 %) and safrole (2 – 20 %) (Nakatanii, 2003).
45
Many other spices are still under investigation for antioxidant potency in food preservation and
health promoting to consumers, following the increasing current demands for natural food and
their health benefits (Dragland et al., 2003). Antioxidant activity screening of both water
soluble and fat-soluble fractions of 1,113 selected food samples at the USDA’s National Food
Nutrient Analysis Program (NFNAP) showed that of the top 50 foods with antioxidant
properties, the best 5 were dried spices, namely ground cloves, dried oregano, ground ginger,
ground cinnamon and turmeric powder (Holversen et al., 2006). When compared with other
categories of food products in the study, herbs and spices displayed the greatest antioxidant
capacity, 0.803 to 125.549 mMol / 100g.
2.17 Assessment of antioxidant activity of antioxidant compounds and the degree of lipid
oxidation in food system
The effectiveness of an antioxidant can be mearsured by quantifying its antioxidant activity or
its ability to delay or suppress lipid peroxidation in biological system. The degree of lipid
oxidation in antioxidant-treated biological systems can be quantified using the following
methods:
1. Measurement of peroxide value (POV) (Sakawa and Matsushita, 1976) using either
iodometry:
ROOH + 2KI ROH + K2 + K2O, or thiocyanide method:
ROOH + Fe2+ ROH + HO* + Fe 3+
2. Measurement of decomposition products (Sakawa and Matsushita, 1976) using any one
of the followings:
46
i. Colorimetry with 2,4 DND derivatives to assay for carbonyl value or with
thiobarbituric acid to assay for TBA value on antioxidant-treated biological system to
determine antioxidant activity,
ii. Gass chromatography to assay for the volatile products to quantify degree of oxidation
in the antioxidant –treated biological system,
iii. GC – MS to assay for the structures of volatile products.
3. Measurement of oxygen consumption (Riely et al., 1974; Gut et al., 1988) usually
achieved by using dissolved oxygen meter on antioxidant-treated biological system to
determine the antioxidant activity.
4. Measurement of double bond by using Infra Red Spectrophotometry method (Gut et al.,
1988) to measure the degree UV absorption in antioxidant-treated biological system.
The ability of any biological system to exhibit antioxidant activity can be quantified
using any of the following method:
1. Measurement of scavenging activity of free radicals such as diphenyl-2-picrylhydraxyl
(DPPH) by the antioxidant using spectrophotometry method,
2. Measurement of reducing power of the antioxidant by assessing its ability to reduce
FeCl3,
3. Measurement of the degree inhibition of linoleic acid peroxidation by the antioxidant
using the Ferric Thiocyanate method,
4. Measurement of oxygen radical absorption capacity of the antioxidant using
spectrophotometry.
47
2.18 Major microorganisms of food poisoning
Food borne pathogens are disease – causing microorganisms transmitted through food during
consumption. Microorganisms are generally contacted from the natural environment during
handling, processing and serving of food. Natural sources of microorganisms include soil, air,
water, plants, animals and their products. Different species of yeast, bacteria and moulds
isolated from raw, processed and cooked foods have been implicated in food spoilage and food
poisoning.
Food borne pathogens that cause most sporadic outbreak of food poisoning include the
infectious ones such as Salmonella and Campylobacter species, and the toxicogenic ones such
as Staphyloccocus aurous and Clostridium botulinum. They have a wide range of minimum
temperature at which they can grow. They also have a wide range of tolerance to thermal
inactivation. Other microorganisms also implicated in food poisoning include Shigella and
Enterococcus species. Microorganisms commonly used as indices of food poisoning include
Listerai monocytogens, Staphylococcus aureus, Salmonella species, Echerichia coli, Bacillus
cereus, Vibrio fulnificus, Clostridium spp, Campylobacter spp and Pseudomonas spp.
2.19 Important preservation techniques for preventing food poisoning from pathogenic
microorganisms
The preservation of food against microbial food poisoning is based mainly on the inactivation
of food-borne pathogens or on the delay or prevention of their growth. Most preservative
techniques manipulate and suppress the physical, chemical and microbial conditions within the
food environment to inactivate microbial growth and survival or totally preclude them from the
food within a time lag. Table 4 summarises the various commonly used preservation
techniques and the mechanism by which foods are protected from poisoning the consumers.
48
Table 4: Categorization of procedures for preserving foods from microbial spoilage
Procedures Factors influencing growth or
survival of microorganisms 1 Cooling, freezing, chill distribution and
storage, frozen storage Low temperature to retard growth
2 Distribution and storage Low temperature and reduction of water activity to prevent growth
3 Drying and curing Reduction in water activity sufficient to delay or prevent growth
4 Vacuum and oxygen-free modified atmosphere packaging
Low oxygen tension to inhibit strict aerobes and delay growth of facultative anaerobes
5 Carbondioxide-enriched modified atmosphere packing
Specific inhibition of some pathogens by carbondioxide
6 Addition of acids Reduction of pH value and sometimes additional inhibition by the particular acid
7 Lactic and acetic acid fermentation Reduction of pH value in situ by microbial action and sometimes additional inhibition by lactic and acetic acids formed, and by other microbial growth
8 Alcoholic fermentation Increase in concentration of ethanol
9 Emulsification Compartmentalization and nutrient limitation within the aqueous droplets water-in-oil foods.
10 Addition of preservatives Inhibition of specific groups of microorganisms
11 Pasteurization and sterilization Delivery of heat sufficient to inactivate target microorganisms to the desired extent
12 Redurization, radicidation and radappertization
Delivery of ionizing radiation at a dose sufficient to inactivate target micro-organisms to the desired extent
13 Aseptic processing Packaging sterilized foods without recontamination
14 Decontamination Treatment of packaging materials and food ingredients with heat, irradiation or chemical agents to reduce microbial contamination.
Source: Gould, (1994)
49
2.20 Antimicrobial properties of spices and spice extracts
From prehistoric time, spices have been used traditionally for medicinal purposes. However, it
is uncertain how and when spices were first used for these purposes. Issues concerned with
food preservation and the prevention of food-borne illnesses have long been an area of concern
to the Scientists, public health officials and the general public. Researchers are continuously
investigating the links between antimicrobial properties of various culinary herbs and spices
and their roles in extending shelf life of food and reducing chronic diseases.
The mechanisms of antimicrobial action by which these spices counteract complications
associated with microbial challenges have been investigated (Lin and Nakano, 1996; Cowan,
1999; Bergonzelli et al., 2003; Okigbo and Igwe, 2007). In counteracting microbial lesion,
spice ingredients can interfere with phospholipid bilayer of the microbial membrane, resulting
in greatest permeability which initiates loss of cellular components and/or impair enzyme
systems needed for production of energy and structural components or inactivate and destroy
the genetic materials of the virulating microbe (Cowan, 1999; De et al., 1999).
Spices which exhibit antimicrobial activity include those that have simple phenols, phenolic
acids, coumarins, terpenes and terpenoids, alkaloids, flavonoids and essential oils as part of
their components (Cowan, 1999; Bergonzelli et al., 2003; Okigbo and Igwe, 2007).
Antimicrobial effects of clove, bay leaves, cinnamon, sage, thyme, oregano, mint, black
pepper, anise seeds, nutmeg and fennel seeds on various food-borne pathogens, including
Salmonella typhimurium,, Staphylococcus aureus, Escherichia coli, Bacillus cereus and vibrio
parahaemolyticus have been reported (Azzaus and Bullerman, 1982; Zaika et al., 1983, Shelef
et al., 1984; Karapinar, 1985; Karapinar and Aktug, 1987; Billing and Sherman., 1998;
Nwuinyi et al., 2009). Also the whole spices, spice extracts, essential oil and essential oil
50
constituents of sage, ginger, rosemary and anise have been shown to confer antimicrobial
activity on many food borne pathogens (Beuchat, 1976; Hitokoko et al., 1980; Shelef et al.,
1984). Conor and Beuchat (1984) showed that out of 32 different plant essential oils screened
for antimicrobial properties, allspice, cinnamon, clove, garlic, onion, oregano, savory and
thyme were popularly inhibitory to selected food spoilage and industrial yeasts. Shelef et al.
(1984) discovered that whole sage inhibited selected food pathogens than sage oil when
incubated in meat broth while in nutrient broth, the sage oil and volatiles were inhibitory than
whole sage. Thus, it cannot be assumed that because an intact spice has traditionally been
considered or even demonstrated to have antimicrobial properties, that its essential oil should
also posses such characteristics. It has been demonstrated that while some essential oils and
plant extracts inhibit the growth of lactic acid bacteria, other plant extracts and essential oils
enhance their growth and survival (Tiwary and Pandey, 1981; Zaika et al., 1983).
Billing and Sherman (1998) noted that countries with hot climates use multiple spices on
regular basis more than countries with cooler climates. Quantitatively, the use of chili pepper,
garlic, onions, anise, cinnamon, coriander, ginger and turmeric is positively correlated with the
mean annual temperature. The reasons for this are not farfetched. Prior to refrigeration, foods
in warmer climates spoil more quickly than those in cold climates. Again, many spices work
synergistically and display increased antimicrobial capacity when used in combinations than
when used in isolation.
2.21 Choice of solvents for preparation of crude extracts from biological materials
The composition of crude extracts from any particular plant material varies with the type and
nature of solvents used for the extraction. Polar solvents tend to extract more of ionic
compounds than non-polar solvents which extract more pure organic molecules. Solvents with
51
low boiling points (extract mainly low molecular weight compounds) leaving behind some
important long chain compounds. Such solvents usually have low latent heat of vapourisation
and require less energy with much ease to be easily removed from the crude extracts after
extraction. Solvents with low viscosity are more mobile and penetrate more easily into the
plant materials to extract the needed components. The choice of solvents for crude extracts
from plant materials depend mainly on the profile of the plant components to be extracted and
the composition of the solvent. Solvents which are inert, immiscible with water, non-toxic and
cheap are most preferred. The solvents must also be of food grade and non-toxic to human.
Table 3 summarises some physical properties of some food grade solvents that are generally
recognised as safe (GRAS) and are commonly used.
Different solvent systems have been used to prepare crude extracts from plant materials such as
spices, fruits, vegetables and other plant parts. Water, aqueous mixture of ethanol, methanol
and acetone are commonly used to extract bioactive compounds from plant materials (Xu and
Chang, 2007). For example, antioxidants were extracted from legumes using absolute water,
absolute methanol, 80 % methanol, 70 % methanol, absolute ethanol, 95 % ethanol, 85 %
ethanol, 80 % acetone, acidic 70 % acetone, 70 % acetone, and 50 % acetone (Xu and Chang,
2005). The yield of bioactive constituents from plant materials depend on the solvent
characteristics, the extraction temperature and chemical composition of the sample. Under the
same condition of extraction time and temperature, the solvent used and the chemical
properties of the spice are two most important factores. There are no documented literatures
that compare the effects of different extracting solvents on the phytochemical composition and
activity of antioxidants and antimicrobials in both the exotic and indigenous spices.
52
Table 5: Some food grade solvents and their physicochemical properties
Solvents Viscosity Lat. heat of
evaporation
(cal. / g)
Boiling
point (OC)
Polarity Allowable
Residue in Food 0 OC 20 OC
Carbon dioxide 0.10 0.07 42.4 -56.6 0.00 TUQ*
Acetone 0.10 0.33 125.3 56.2 0.47 TUQ
Ethanol 0.77 1.20 204.3 78.3 0.68 TUQ
Hexane 0.40 0.33 82 68.7 0.00 1PPM
Methanol 0.82 0.60 262.8 64.8 0.73 TUQ
Dichloromethane - 0.43 78.7 40.8 0.32 0.1PPM
Pentane 0.29 0.24 84 36.2 0.63 1PPM
Propan-2-ol - 2.43 167 82.3 0.63 1PPM
Water 1.80 1.00 540 100.0 0.73 TUQ
Propylene glycol - 56.00 170 187.4 0.73 TUQ
Glycerol 12110.0 1490.0 239 290 0.73 TUQ
Source: Moyler, 1988. *TUQ = Technical unavoidable quantity
53
2.22 Biology and ecology of Tetrapleura tetrapetra (Schum & Thonn)
Tetrapleura tetrapetra has two varieties, namely Tetrapleura thoningii and Adenonthera
tetraptera, both of which belong to the Genera Tetrapleura and of the family Minosaceae. The
plant is a perennial tree, about 30 m high and found in the lowland forest of tropical Africa
particularly in the West Central and East Africa.
The fruit consists of a fleshy pulp with some small brownish black seeds. The fruits are green
when tender but dark, reddish brown when fully ripe. The fruit has four longitudinal wing-like
fleshy ridges of about 10 cm broad with two of the ridges hard and woody while the remaining
two are soft and fluffy. The fruits have fragrance, pungent aromatic odour.
2.28 Nutrient composition of Tetrapleura tetrapetra
The shells, pulps and seeds of both fresh and dry fruits of Tetrapleura tetrapetra have
varying amounts of protein, lipids and minerals, all of which are comparable and in some cases
even higher than those of popular spices such as red pepper, onion, curry and ginger ((Essien et
al., 1994; Okwu, 2003). Crude protein is very low in the fleshy mesocarp (2.12 %) and seed
(0.51%) but is not at all in the woody mesocarp (Essien et al., 1994). Both fresh fruits and
seeds are rich in potassium, iron, magnesium and phosphorus but low in sodium. The fruits
have less than 5 mg / 100 g of zinc and nickel. Sucrose and fructose occur in traces in both the
fruits and seeds.
The dried fruits have 7.44 % to 17.50 % crude protein, 17.0 % to 20.24 % crude fibre, 4.98 to
20.36 % lipid, 43.18 % to 49.06 % carbohydrate and 234.42 g / cal to 379.48 g / cal food
energy (Okwu, 2003). The fruit oil is a good drying oil with a few unsaturated bonds. The
54
seeds have the amino acids L-r- methylene glutamic acid and L-r- ethyldiene glutamic acid
(Gmelin and Olesen, 1967). The fruit contains cinnamon and caffeine acids (Adesina et al
1980). The fruit also contains the essential oils saponosides triterpenes, - aessculetin,
caumarins, tannins, steroids and triterpene glycosides. Also present in the fruit are the
phytochemicals, oxalates (8.14 to 16 Mg / 100 g), tannins (16.5 to 35.7 mg / 100 g) and
hydrocyanic acid (hydrogen cyanide) (98 to 100 mg/100 g), saponins, alkaloids, steroids and
flavonoids.
2.29 Food and medicinal uses of Tetrapleura tetrapetra
The fruits are used locally in Nigeria for food flavouring, in soap and pomade preparations.
The fruit is premixed with soap base made from palm kernel oil or shear butter to improve the
foaming properties of the soap (Adebayo et al., 2000). Convalescents bathe with the infusion
of the fruit for healing. The bark, root and fruit of the plant are used in the management of
convulsion, leprosy, inflammation and rheumatic pains (Dalziel, 1948; Adesina et al., 1980).
Infusion of the whole fruit is taken as a recuperative tonic (Ojewole and Adesina, 1983).
Saponin extracted from the fruits has been proven to be a potent hypotensive and
nonsposmolytic agent in traditional infusion (Obidoa and Obasi, 1991).
In Eastern Nigeria, the fruits are used to prepare pepper soups for mothers after labour to
prevent postpartum contraction (Nwawu and Akah, 1986), and also for normal cooking for its
flavouring and cleansing effects. Extracts of the fruits exhibit anti-ulcer activity, confirming
its use in ethnomedical medicine to treat ulceration in gastro-intestinal disorder (Noamesi et
al., 1992).
55
2.30 Ecology, botany and distribution of Monodora tenuifolia (Benth)
Monodora tenuifolia (Benth) (Annonaceae) is a shrub that grows into a medium size forest
tree. It is variously known as African nutmeg (English) (Burkill, 1985) and ehuru ofia (Igbo),
and found in the forest zones of East Indies, West Indies, Sri Lanka, Africa and Malaysia
(Talaji, 1965). In Africa M. tenuifolia is widely distributed along the West Coast (Adeoye et
al., 1986) and very common in Nigeria, Ghana, Cameroon, Gabon and Zaire (Congo
Democratic Republic) where it is used as an ornamental plant, flavouring ingredient and as an
active ingredient of many traditional medicines (Burkill, 1985).
2.31 Chemical composition and uses of Monodora tenuifolia (Benth)
Proximate analysis of the seeds from different sources revealed variable contents of
carbohydrate, Protein, fats and oil, crude fibre and minerals (Burkill, 1985; Ezenwali et al.,
2010). The seeds have high oil content (34.7 to 68.8 %), with triacylglycerol being the
dominant lipid group in the oil. Unsaponifiable lipids occur in low concentrations. The seed
extract has been shown to contain the phytochemicals alkaloids, flavonoids, saponins, sterols
and terpenes (Ezenwali et al., 2010). The volatile components of the fruit essential oil are
mainly sesquiterpene hydrocarbons, a mixture of monoterpenoid and sesqueterpene
(Adesomoju et al., 1991).
The fruits are used as spice for food flavouring (Irvine, 1961; Ogutimein et al., 1989). The
fruits are aromatic and are used as ingredients of many herbal medicines in Southern Nigeria.
The fruits, bark and leaves are widely used in traditional medicine to treat tooth ache,
dysentery, dermatitis and headache (Nielson, 1979; Adeoye et al, 1986).
When roasted, the ground seed is rubbed on the skin to cure skin rashes (Irvine, 1961).
56
2.27 Ecology, botany and distribution of African nutmeg (Monodora myristica Gaetn)
Morphologically, African nutmeg (Monodora myristica) is a perennial edible plant of the
Annonaceae family. It is a berry that grows wild in evergreen forests of Africa (Burubai et al.,
2008). It is an ornamental tree of up to 30 m high, with dense foliage. The stem is flutted; the
outer bark is thin and dark brown while the inner bark is light brown above and pale cream
beneath. The stem is aromatic. The leaves are elliptical, sometimes becoming wider at the
apex, about 14 – 15 cm long and 5 – 14 cm broad, and arranged alternately.
The plant flowers between September and April, at the time of appearance of new leaves. The
flowers are large, fragrant and pendant, hanging on very long stalk with a crinkly bract of
about 2.5 cm long near the end of the stalk. The sepals are about 4 cm long, spotted with red
wavy edges and crisped. The flower contains about 6 petals, with the outer ones about 10 cm
long, brightly yellow coloured and with dark red mark on the edges. The inner petals are sub
triangular in shape, dull cream yellow in colour with red sports in the inner side.
2.28 Chemical composition and uses of African nutmeg (Monodora myristica Gaertn)
The fruits are produced between April and September. They are about 15 cm in diameter,
green, round, and usually suspended in large stalk. The pulp is white and contains numerous
seeds. The seeds are composed of moisture (14.7 %), protein (9.1 %), oil (29.1 %), food energy
(458 kcal / 100 g), fibre (25.9 %) and ash (2.3 %) (Burubai et al., 2008). The seeds are also
rich in potassium, phosphorus, calcium and magnesium.
The seeds yield colourless, volatile oil with a pleasant taste and odour. The seeds are used as
condiments for soups, and are added into snuff as flavouring agent. The seeds are applied
57
externally to treat migraine, taken orally as a stimulant for stomach cleanser, caminative and
antiparasitic medicine as well as treatment for guninea worm (Agoha, 1974)
2.29 Ecology, botany and distribution of Ocimum viride (Willd)
Ocimum viride (Labiatae), also called Ocimum gratissimum L is an erect, small shrub with
many branches and is usually not more than 1m high (Iwu, 1993). It is called ‘nchuanwu’ in
Igbo, efinrin in Yoruba and ‘dai doyata gida’ in Hausa. The leaves are simple, up to 9 cm long
and 4.5 cm broad with unequal sided base and toothed margin. The leaves are sparsely hairy on
the undersurface and pitted with glands. The flowers are creamy white or yellowish in colour
and are about 12 cm long. The flowers occur as small 4 – lobed capsules (Parry, 1969). The
plant occurs in the deciduous forest and savanna zones of the tropics around village–huts and
gardens. It is cultivated mainly for medicinal and flavouring purposes.
2.30 Chemical composition and uses of Ocimum viride
The leaves have very aromatic volatile oil that contains mainly thymol and eugenol. The leaves
also contain xanthones, terpenes and lactones (Farrel, 1990). Thymol isolated from the plant
has been shown to be antiseptic and antispasmodic (Iwu, 1993).
In Nigeria, the spice volatile oil has been shown to exhibit antimicrobial, insect repellant, and
antihelmintic activities (Farrel, 1990; Iwu, 1993). Oral and topical formulations from the plant
have been evaluated in Nigeria. The whole herb is used throughout West Africa as an
ingredient of many malaria remedies and the leaf oil is used to prevent mosquito bites. The
crushed leaves are instilled into the eye for treating conjunctivitis while the oil from the leaves
is used for dressing wounds and mouth gargle. The leaves are also used in Nigeria to cure
stomach ache and catarrh. The leaf extract is used as a general tonic and anti diarrheal. The oil
58
is mixed with ethanol and used as lotion for skin infections and taken orally for bronchitis
(Farrel, 1990; Iwu, 1993).
59
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Materials
The four spices, Tetrapleura tetrapetra (Schum &Thonn) (Essien et al., 1994), Monodora
myristica (Gaertn) (Burubai et al., 2008), Monodora tenuifolia (Benth) (Burkill, 1985) and
Ocimum viride (Willd) (Iwu, 1993) were purchased from Nsukka in Enugu State, Nigeria.
Spices were transported to the Food Science and Technology laboratory, Department of Food
Science and Technology, University of Nigeria, Nsukka where they were stored at ambient
temperature (26 ± 2 OC) until used.
Fresh beef and pork (thigh muscle), vegetables (spinach and pumpkin) and parboiled rice were
purchased from Ikpa market, Nsukka, Nigeria. The rice, vegetable mix (1 : 1 ratio of spinach
and pumpkin) and part of the beef and pork were used for anti-microbial screening. The
remaining part of the beef and pork were used for antioxidant activity screening of the spices.
The vegetables were refrigerated (4 OC) while the beef and pork were frozen (about -18 OC)
overnight and thawed the following day for preparation for the antimicrobial and antioxidant
screenings.
3.2 Preparation of spice extracts
The spices Monodora tenuifolia (seeds), Monodora myristica (seeds), Ocimum viride (leaves)
and Tetrapleura tetrapetra (fruits) were sun-dried for 72 h. The Monodora myristica seeds
were toasted (100 – 120 OC for 15min) and cracked to recover the nibs. Each of the spices was
60
ground into coarse particles using Hammer mill (Betsch 5657 GmbH, Germany) and 2.5 g of
each was homogenized with 100 ml of the appropriate solvent [distilled water, 95 %
methanol, acetone / hexane (1:1,v/v), n-hexane / methanol / acetone (2:1:1, v/v/v), acetone /
water / acetic acid (70:29.5:0.5, v/v/v)] for 3 min in sterile, tightly corked 100 ml bottles. After
homogenization, the bottles were rested for 3 h, re-homogenized for another 2 min and filtered
through muslin cloth into 100 ml bottles.
Extracts were tightly corked and then boiled for 5min. in water baths to inactivate inherent
enzymes (Effraim et al., 2000). After cooling under tap water, the crude extracts were further
filtered through double-walled whatman no 5 filter papers, concentrated to dryness in a rotary
evaporator (Model type 349 / 2, Corning Ltd.) at 70 OC, then tightly corked in sterile bottles
and stored at refrigeration temperature (4 OC) in the dark for use within two days. Required
concentrations [4, 8, 12, 16, 20 and 25 mg of dry matter/ml (D.M/mL)] were prepared from the
crude extracts by reconstituting with appropriate solvents and then used to determine
phytochemical compositions and antioxidant properties of the spices.
3.4 Preparation and storage of cooked ground beef and pork patties
Each of the four dry spices, namely Tetrapleura tetrapetra, Monodora myristica, Monodora
tenuifolia and Ocimum Viride, was milled into small particle sizes using a kitchen grinder and
then sieved through 160 µm mesh sieve to get fine powder. Each spice powder was divided
into three levels of 1.2g, 2.4g and and 4g and used to prepare the meat. Freshly cut beef thigh
muscles (5 kg) and pork (4.5 kg) were separately boned manually with kitchen knives, and the
free muscles diced into small pieces (about 1cm x 1 cm x 1 cm) before being minced in a
mechanical meat mincer. The minced muscle was weighed into 17 portions of 200g each for 4
(spice treatments) X 4 (spice levels). Four spice levels (0.0 %, 0.6 %, 1.2 % or 2.0 % w/w meat
61
weight, i.e., 0.0, 1.2, 2.4 and 4.0 g in 200 g of meat) were admixed separately with 5 ml of
distilled water, and blended appropriately with the 200 g of beef or pork. The untreated
samples (no spice) served as the (negative) controls. The lowest (0.1 %) to highest (2.0 %)
levels of the spices in this study were estimated from the various levels of household use of the
spices (Dwivedi et al., 2006).
The blends were thoroughly cooked (100 to 105 OC) for about 30min. using a gas cooker. The
cooked meat samples were divided into two portions, and each portion formed into patties in
100ml re-sealable plastic plates and then cooled for about 30 min at room temperature (26 ± 2
OC). The plates and contents were then sealed with lids and stored at 4 OC for 16 days.
3.5 Proximate analyses
Moisture, crude protein, fat, ash and fibre contents were determined in triplicates by the
Official Methods of AOAC (2000).
3.4.1 Moisture content
Moisture content was determined by difference in weight after heating a weighed sample, in a
hot air oven at 105 OC for 4h according to the method of AOAC (2000). Ground spice sample (
3 g ) was placed in a weighed (W1) oven-dried porcelain dish. This was reweighed to get the
weight of porcelain and spice sample (W2), and then dried in a hot air oven at 105 OC until a
constant weight (W3) was obtained on cooling.
Moisture content was calculated with the expression:
%Moisture = W2-W3 /W2-W1 x 100 / 1.
62
3.4.2 Crude protein content
Ground sample of spice ( 2 g), a Kjeldhal tablet (containing 20 g K2SO4, 1 g CuSO4 and 0.05 g
Selenium), 25 ml concentrated tetraoxosulphate vi acid (H2SO4) and about 5 pieces of glass
beads (to prevent bumping) were put in a Kjeldhal flask. Each sample was digested (heated) in
a fume chamber, first gently and then vigorously to about 420 OC with intermittent shaking for
about 30 min. until a green solution resulted. The solution was cooled and then diluted to 250
ml with distilled water. Water was distilled through Markham distillation apparatus of Micro
Kjeldhal distillation unit for about 15 min to get distillate for blank titration.
About 5 ml of the resulting digest solution and 5 ml of 60 % sodium hydroxide (NaOH)
solution were separately pipetted into the Markham distillation apparatus. The resulting
mixtures were then steam-distilled for about 10 min to collect enough ammonium sulphate as
distillate down a condenser dipping inside 5 ml boric acid and indicator solution in a 100 ml
conical flask. The boric acid plus the indicator solution changed colour from red to green
showing that all the ammonia liberated has been trapped. The distillates (Vml) were separately
titrated with 0.01 M HCl until a sharp colour change persisted. The nitrogen (% Nitrogen)
content and crude protein content (% crude protein) were calculated with the expression:
% Nitrogen = (Vs – Vb) x Macid x 0.01401 x (250/5) x 100 / W;
% Crude protein = % Nitrogen x 6.25, after determining nitrogen content by wet digestion
analysis; where: Vs = Vol (ml) of acid required to titrate sample, Vb = Vol (ml) of acid required
to titrate blank, titre value = Vs - Vb, M = molarity (0.01 M) of acid used, Total volume of
digest = 250, Volume of digest used for distillation = 5, Atomic mass of N = 0.0140, W =
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Weight of sample in grams (2 g), Dilution factor = (250/5) and 6.25 = conversion factor (100 /
% N in food protein) for most plant food and mixes.
3.4.3 Ether extract
Ether extract was determined according to the method of AOAC (2000), using soxhlet
extraction unit (All-Clevenger apparatus). A clean 250 ml boiling flask was dried in a hot air
oven, cooled in a desicator and then filled with about 200 ml of petroleum ether (boiling point
40 -60 OC) before fixing it back to the soxhlet extraction unit. A known weight, W1 ( 3 g) of
ground spice sample was placed in a soxhlet thimble which was then plugged with light cotton
wool, weighed, W2 and then fixed in place in the soxhlet extraction unit. The soxhlet extraction
unit was then assembled and allowed to reflux for about 6 h, after which the thimble was
carefully removed from the unit. Petroleum ether was recovered through the upper arm of the
unit. The thimble was dried at 105 OC for 1h, cooled and then weighed to get the weight, W3,
of thimble and defatted food sample.
% Ether extract = W2 – W3 / W1 x 100/ 1= .weight of fat/weight of food sample x 100/1.
3.4.4 Crude fibre content
Crude fibre was determined according to the method of AOAC (2000). Defatted spice sample
(2 g) was dried in a desicator; mixed with 200 ml of 0.225 M tetraoxosulphate vi acid and
some antifoaming agents in 500 ml beaker. The beaker was covered with watch glass and
boiled for about 30min, after which any loss in volume was made up with distilled water. The
resulting solution was filtered through several layers of muslin cloth on fluted funnel and the
residue washed with boiling water until acid free. The residue was then transferred into another
beaker containing 200 ml of 1.25 g of trioxocarbonate-free NaOH per 100 ml solution and
boiled for 30 min. This was filtered through Buchner’s funnel lined with weighed what man no
54 filter paper, ensuring that all the residue was washed with hot boiling distilled water until
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alkaline free. The residue was allowed to drain, transferred to weighed porcelain and dried at
105 OC for 4 h. The dried residue was then incinerated and weighed to calculate % Crude fibre.
% Crude fibre = Loss in weight after incineration x 100.
3.4.5 Total ash content
Ash content was determined according to the method of AOAC (2000). Finely ground spice
samples (1 g for each) were oven-dried at 105OC for about 4 h. The dried sample (W2) was
charred and incinerated in a preheated muffle furnace at 550 OC for about 12 h or until light
grey or white ash of constant weight, W3 resulted. The ash content was calculated with the
expression:
% Ash content = Weight of ash / Weight of original food sample x 100 /1.
3.5 Energy value
Ground food samples (25 mg) were pelleted and ignited in a Gallenkamp Autobomb automatic
adiabatic oxygen bomb calorimeter at 25 ATP. This oxidized food caused a rise in temperature
(t OC) of surrounding water in the calorimeter, and the temperature increase was used to
estimate the energy value of the food. The length (L) of wire consumed from the ignited wire
was estimated. The heat of combustion of the food expressed as gross energy was calculated
using the expression:
Gross energy = (WxT-2.3L-V)/G, where W = Energy equivalent of calorimeter,, T = Temperature rise, 233 = Constant heat of combustion of wire,, G = Weight of sample in grammes, V = Volume of gas generated. 3.6 Digestion and analysis for minerals
Analysis for Sodium, Potassium, Calcium, Iron, Magnesium, Selenium and Zinc was carried
out after wet digestion using the method of AOAC (2000). Ground samples (0.5 g) of each
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spice was boiled (100 OC) with 5 ml concentrated nitric acid (HNO3) and 5 ml of 30 %
hydrogen peroxide (H2O2) solution continuously for about 2 hours in an electric heating
mantle (HP 220, LITEC Product Inc. Albany, N-Y., USA) until clear solutions were obtained.
These were cooled, filtered through Whatman no 45 filter papers and then through < 0.45
millipore filter papers. Filtrates were made up to the 25-ml mark of the volumetric flasks with
distilled water and then used to analyse for the individual minerals using Atomic Absorption
Spectrophotometer (Buck Scientific AAS Model 210, equipped with single slot burner and air-
acetylene flame).
Preparation of standards for analysis of minerals in samples
Working standard solutions of sodium, potassium, calcium, iron, magnesium, selenium and
zinc were prepared from the stock standard solutions containing 1000 ppm of each element in
2N nitric acid solution. Calibration and measurement of absorbance of each element against a
blank at its unique wavelength was done using Atomic Absorption Spectrophotometer (A.
Analyst 300, Perkin Elmer, Morwalk, Conn, U.S.A). The calibration curves were prepared
separately for each element. Absorbance of each element in the filtrate was read at its
wavelength from the spectrophotometer and its concentration in the spice extrapolated from the
standard curve.
Total phosphorus content in each sample was determined spectrophotometrically by the
phosphovanadomolybdate method (AOAC, 2000). Phosphorus stock standard containing 2000
g of phosphorus per litre of stock standard solution was prepared by dissolving 1.1224 g of
dibasic potassium phosphate (K2HPO4; M. Wt. = 174) in 500 ml deionized water, acidified
with 8ml concentrated HCl before diluting to 1000 ml in a 1-litre volumetric flask. A 25 ml
portion of the stock standard was diluted to 100 ml with 10 % trichloroacetic acid solution to
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give a working solution which was further diluted to concentrations (P) of 0.005 mg / ml, 0.10
mg / ml, 0.15 mg / ml, 0.20 mg / ml and 0.50 mg / ml. The absorbance values were read from
an Atomic Absorption Spectrophotometer (A. Analyst 300, Perkin Elmer, Morwalk, Conn,
U.S.A) at 660 wavelengths and then plotted against the concentrations to produce the standard
curve from which the concentration of phosphorus in the samples at known absorbance was
extrapolated.
3.8 Determination of vitamins
3.7.1 Determination of ascorbic acid (Vitamin C) content
The ascorbic acid content of the samples was determined by the method of AOAC (2000).
Ground spice (1 g each) were homogenized with 50 ml of distilled water for 3 min, rested for 3
h, re-homogenized for another 2 min and filtered through muslin cloth.
Extracts were tightly corked and then boiled for 5min. in water baths to inactivate inherent
enzymes (Effraim et al., 2000). The samples were cooled under tap water and filtered through
double-walled whatman No 5 filter papers into tightly corked, sterile bottles. Ten (10) ml of
each spice extract was mixed with 25 ml of 20 % glacial acetic acid and titrated against
standardised 2, 6–dichloro indophenol (0.05 g / 100 ml) solution. Ascorbic acid was used as a
standard, and the concentration of ascorbic acid in the samples was calculated and expressed as
mg/100 g of the dry spice sample.
Mg ascorbic acid / 100 g = C x V x (F/W)
Where, C= mg ascorbic acid/ml 2,6-dichloroindophenol used,, V= volume of 2,6-dichloroindophenol used,, F= dilusion factor,, W= weight of sample used. 3.7.2 Determination of niacin content
Niacin content was determined according to the method of Eitenmiller and DeSouza (1985).
Ground spice sample (50 g) was admixed with 200 ml of 1N H2SO4 to extract niacin. This was
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autoclaved at 121 OC for 30 min, cooled and the pH adjusted to 4.5 before diluting to 250 ml
mark. The mixture was filtered and purified by pipetting 40 ml of filtrate into a 50 ml
volumetric flask containing 17 g (NH4)2SO4, shaken and then filtered. To develop the colour, 1
ml of each filtrate and standard solution were pipetted into two separate test tubes. Then, 0.5
ml of 2 % aqueous NH4OH, 2.0 ml of 2 % H2SO4 solution and 0.5 % of dilute HCl were added
to each tube and mixed vigorously. Next, 5.0 ml of H2O was added into the spice filtrate while
5.0 ml of CNBr was added into the niacin standard; and both shaken vigorously. Both mixtures
were allowed to stand for 2 min after which absorbance was read against a standard at 430 nm
and niacin content extrapolated from niacin standard curve.
3.7.3 Determination of riboflavin content
Riboflavin content was determined according to the method of AOAC (2000). The spice was
ground and 5 g of it mixed with 50 ml of 0.2N HCl in a 100 ml conical flask, boiled for 1h and
cooled under tap water. The pH of the mixtures was adjusted to 6.0, using 0.5 M NAOH
solution and then readjusted to 4.5 using 1N HCl to facilitate precipitation of all interfering
materials. It was diluted to the 100 ml mark of flask and then filtered through a double-fold
filter paper.
Ten (10) ml of the filtrate was added to each of four separate test tubes. To each of first two
test tubes was added 1ml of distilled water while to each of the remaining two test tubes was
added 1ml of riboflavin standard (0.5 µg / ml). One (1.0) ml glacial acetic acid and 0.5 ml of
3% KMnO4 was added to each of the tubes and the tubes were shaken vigorously.
Fluorescence was measured at 440 nm extincsion and at 565 nm emission for the sample tube
containing water, and then repeated on the same sample after admixing with 20 mg of
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Na2S2O4. Fluorescence of standard at same 440 nm excitation and 565nm emission was also
measured. Riboflavin concentration was calculated with the expeesson:
µg Riboflavin / g sample = [(A – C) / (B – A)] x (S / V) x (F / W)], where,
A = Fluorescence of sample containing water,
B = Fluorescence of sample containing riboflavin standard,
C = Fluorescence of sample containing Na2S2O4,
S = Concentration of standard (µg / ml),
V = Volume of sample extract used for fluorescence measurement,
W = Weight (g) of sample used,
F = Dilution factor.
3.7.4 Determination of thiamin content
The method of AOAC (2000) was used to determine the thiamin content in the spices. Five
grams of each of the spices were homogenised with ethanolic sodium hydroxide (50 ml). Each
homogenate was filtered into a 100 ml flask, and 10 ml of the filtrate pipetted into a test tube to
which 10 ml of potassium dichromate was added to develop colour. A blank sample was
prepared and the colour also developed. Absorbance of samples was read at 360 nm. A
standard solution was prepared using thiamic acid to get 100 ppm and serial dilutions of 0.0,
0.2, 0.4 and 0.8 ppm was made. This was used to plot a calibration curve from which thiamin
contents of the spices were extrapolated using the absorbance values.
3.8 Determinations of phytochemical composition
3.8.1 Determination of total phenol
Total phenol content was determined using Folin-ciocalteau method (Roesler et al., 2006).
Folin-ciocalteau method allows the estimation of all flavonoids, anthocyanins, and
nonflavonoid phenolic compounds, including phenols and tannins, (that is, all phenolics
present in the sample) (Roesler et al., 2006). The total phenol content of the various spices was
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determined by mixing 0.5ml aliquot of freshly prepard sample extract with equal volume of
water, 0.5 ml Folin-Ciocalteu’s reagent, and 2.5 ml of saturated solution of sodium carbonate
(Na2C03). The absorbance was measured after 40 min at 725 nm (Singleton et al., 1999).
Garlic acid was used at concentrations of 0.0, 3.0, 6.0, 12.0, 18.0, 24.0 and 30.0 µg / ml to
prepare total phenol standard curve. Total phenol content was extrapolated from the standard
curve using the absorbance values and expressed as garlic acid equivalents (GAE / 100 g).
3.8.2 Determination of total flavonoid
The Iron (iii) Chloride (AlCl3) method of Lamaison and Carnet (1990) was used to determine
the total flavonoid contents in the spice sample extracts. Aliquot of 1.5 ml of each freshly
prepared spice extract was added to equal volumes of a solution of 2 % AlCl3.6H20 (2 g in 100
ml methanol). The mixture was shaken vigorously, and absorbance read at 367 nm after 10
min of incubation. Garlic acid was used at concentrations of 0.0, 0.02, 0.05, 0.10, 0.50, 1.50
and 2.0 mg / ml to prepare a standard curve for flavonoid. Flavonoid content was extrapolated
from the standard curve using the absorbance values and expressed as garlic acid equivalents
(GAE / 100 g).
3.8.3 Determination of condensed tannin content
The condensed tannin content of the various spices was determined according to the method
of Price and Butler (1977). The freshly prepared extract (1 ml) of each spice was diluted to 10
ml with distilled water, and mixed with 0.5 ml of 0.1 M FeCl3 in 0.1 NHCl and 0.5 ml of
0.008 MK3Fe (CN)6. The mixture was allowed to stand for 1 min for colour development, and
absorbance was read at 720 nm. Tannin content was extrapolated from a standard curve
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(prepared with tannic acid at concentrations of of 0.0, 0.01, 0.04, 0.08, 0.15, 0.20, 0.50 and 1.0
mg / ml) and expressed as tannic acid equivalents (TAE / 100g).
3.9.4 Determination of total anthocyanin content
The total anthocyanin content in the spice extracts was determined using the pH differential
method of Fuleki and Francis (1968) as described by Glusti and Wrolstad (2001), with a slight
modification. The freshly prepared extract of each spice was divided into two equal parts of
about 10ml each; and their pH values adjusted either to 4.5 by adding 1N HCl or to 1.0 by
adding 12N HCl. The absorbance of the two samples at the pH of 4.5 and 1.0 was measured at
520 nm and 700 nm respectively, using the UV visible spectrophotometer (A. Analyst 300,
Perkin Elmer, Morwalk, Conn, U.S.A). The anthocyanin content of the samples was calculated
in milligrams Cyanidin-3-glucoside per gram of absolutely dry matter of the spices, using
molar extinction coefficient of 26900 and molecular weight of 449.2.
3.9.5 Determination of carotenoid content
Carotenoid content was determined using the method AOAC (2000). One gram of ground
spice was mixed with 10 ml of acetone in a 50 ml conical flask and allowed to rest for 20 min.
with intermittent gentle shaking every 4 min. After sedimentation, the upper clear layer was
decanted into a clean text tube and 5 ml of benzene added. This was shaken vigorously and the
upper layer separated using a separating funnel. The absorbance read at 453 nm and used to
extrapolate carotrdenoid content from a standard curve.
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3.9.6 Determination of alkaloid content
The alkaloid content was determined using the gravimetric method of Harborne (1973). The
ground spice samples (5.0 g) were each dispersed in 50 ml of distilled water, 95 % methanol,
acetone / hexane, n-hexane / methanol / acetone and acetone / water / acetic acid solvents in
250 ml volumetric flasks. These were shaken vigorously and allowed to rest for 4 h before
being filtered through Whatman no. 5 filter paper.
The filtrates were then evaporated to one quarter (1/4) of original volumes, after which
concentrated ammonium hydroxide (NH4OH) was added drop-wise to each alkaloid until
precipitate persisted. The mixtures were then filtered through weighed filter paper, and the
alkaloids residues washed with 1 % ammonium hydroxide solution. The filter papers and
contents (alkaloids) were oven-dried at 60 OC for 30 min. and reweighed to determine alkaloids
contents using the expression,
Percentage (%) alkaloid = w2 – w1 X 100 W 1 Where, w = weight of spice sample, W1 = weight of empty filter paper, W2 = weight of filter paper plus alkaloid precipitated.
3.9.7 Determination of phytate content
Phytate content was determined according to the method of AOAC (2000). The spice sample
(4.0 g) was soaked in 100 ml of distilled water, 95 % methanol, acetone/water/acetic acid,
acetone/hexane or hexane/methanol/acetone solvent for 3 h and then filtered through Whatman
N0. 2 filter paper. The filtrates (25 ml) were pipetted into 50 ml conical flasks, and 5ml of 0.3
% ammonium thiocyanate solution added, after which 53.5 ml of distilled water was added and
the mixtures were titrated against standard Iron (iii) Chloride solution containing 0.00195 g
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Fe3+ / ml until a brownish yellow colour persists for 5min. The phytate content of the spices
was expressed as percentage (%) phytate in the spice sample.
3.9.8 Determination of oxalate content
Oxalate content of samples was determined as described by Oke (1966). A blend of each
ground spice sample (1.0 g) in 190 ml of distilled water, 95% methanol, acetone/water/acetic
acid, acetone/hexane or hexane/methanol/acetone solvent and 10 ml of 6M HCl in 250 ml
volumetric flask was digested in a water bath at 90 OC for 4 hours, and then centrifuged at
2000 rpm for 5 min. The supernatant was diluted to 250 ml with distilled water; and then
titrated with concentrated ammonium hydroxide solution dropwise, using methyl orange as an
indicator.
Titration was done to determine endpoint when the pink colouration changed to faint yellow
colour. The resulting solution was heated at 90 OC for about 20 min. on a water bath and 10ml
of 5 % Calcium Chloride (CaCl2) solution was added to precipitate oxalate as Calcium oxalate.
The resulting solution was rested overnight, then centrifuged and decanted to get the residue.
The residue was oven-dried at 60 OC for 48 h, cooled and then weighed. This was repeated
thrice and the mean weight determined and expressed as percentage oxalate content using the
expression,
% Oxalate content = weight of oxalate / weight of spice sample x 100 /1.
3.9.9 Saponin content determination
Ground sample (1 g) of each spice was macerated in 10ml of distilled water, 95 % methanol,
acetone/water/acetic acid, acetone/hexane or hexane/methanol/acetone; and the extract decanted into
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a 50 ml beaker. The residue was re-extracted with another 10 ml of solvent, allowed to rest and
then decanted into the formal beaker. The extract was pooled together and evaporated to
dryness, re-dissolved in 6ml of ethanol and 2 ml of the ethanolic extract allowed to stand for 30
min for colour development. Absorbance was read at 550 nm and used to extrapolate saponin
content from a standard curve.
3.10 Determination of antioxidant Properties of Spices
3.9.1 Determination of free radical scavenging activity
Free radical scavenging activity of the spice extracts was determined using the radical 1, 1-
diphenyl-2-Picrylhydrazyl (DPPH), which is widely used to evaluate the free radical
scavenging activity of natural antioxidants (Brand–Willams et al., 1995; Bondet et al., 1997;
Sanchez- Moreno et al., 1998). A 1000 µL volume of the spice supernatant was mixed with
1000 µL of 0.4 M DPPH radical in ethanol solvent (0.004 % W/V). The mixture was left in the
dark for 30 min before reading the absorbance at 517 nm. Radical scavenging was expressed as
the inhibition percentage and was calculated using the formula of Yen and Duh (1994)
% Inhibition = [(ADPPH – AEXTRACT) / ADPPH] x 100, where ADPPH = absorbance of DPPH
radical at 517nm and AEXTRACT = absorbance of extract of spice at 517 nm.
3.9.2 Measurement of reducing power of the crude extracts of spices
Reducing power of the crude extracts of spices was determined according to the method of
Yen and Chen (1995). The crude extracts (5ml) of spice or BHT (5 ml) were separately mixed
with equal volume of 0.2 M phosphate buffer (pH, 6.6) and 1 % potassium ferricyanide. The
mixture was incubated at 50 OC for 20 min, after which an equal volume of 1 % trichloro acetic
acid (TCA) was added to the mixture and then centrifuged at 3000 g for 10 min. The upper
74
layer (the supernatant) of the suspension was mixed with distilled water and 0.1 % FeCl3 in the
ratio of 1:1:2, and the absorbance measured at 700 nm. Increased absorbance of the reaction
mixture indicated increased reducing power.
3.9.3 Determination of antioxidant activity of crude extracts of the spices by the Ferric
thiocyanate (CTC) method
The ferric thiocyanate (FTC) method was adopted from Osawa and Namiki (1981) method.
The spice crude extracts (2.5 ml) were added to 2.5 ml of 95% (V/V) ethanol, and then mixed
with 4.1 ml of linoleic acid (2.51 % V/V) in 99.5 % (V/V) ethanol, 8ml of 0.05 M phosphate
buffer ( pH 7.0), 3.9 ml of distilled water and then kept in the dark in screw- capped containers
at 4 OC. To 0.1 ml of this solution was added 9.7 ml of 75 % (V/V) ethanol and 0.1 ml of 30 %
(W/V) ammonium thiocyanate. A 0.1 ml volume of 20 mM Ferrous chloride in 3.5 % (V/V)
hydrochloric acid was added to the reaction mixture, and the absorbance of the resulting red
solution measured after 3 min at 500 nm repeatedly at interval of 24 h until the control (no
extract) reached the maximum value. This was run in triplicates and results averaged.
The percentage inhibition of linoleic acid peroxidation was calculated as:
(%) Inhibition = 100 - {Absorbance increase of sample X 100) (Absorbance increase of blank}
3.10 Determination of antimicrobial properties of the spices.
3.10.1 Minimum inhibitory concentration (MIC) determination
The MIC (minimum inhibitory concentration) of Tetrapleura tetapetra, Monodora myristica,
Monodora tenuifolia and Ocimum viride was determined using the aqueous and ethanolic
extracts of the spices as described by Kim et al. (2006). The concentrated extracts were
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reconstituted with heat-sterile phosphate buffered Saline (PBS) broth to the desired
concentrations of 4, 10, 20, 30, 40 and 50 mg / ml. Media at the desired test concentrations
were inoculated with microorganisms to yield initial numbers between 104 to 106 cells / ml,
and incubated at 37 OC for 24 h. The sensitivity of the test organisms was expressed as the
MIC (mg / ml) of each extract. Experiments were conducted in quintuplicate, and the lowest
value of extracts (mg / ml) showing complete inhibition was recorded as the MICs; a complete
absence of growth based on the viable count after the incubation period was regarded as no
growth (Man and Markham, 1998).
3.10.2 Preparation and inoculation of substrates
The culture medium used was nutrient broth while the foods from which extracts were
prepared included fresh beef, parboiled rice and fresh vegetable mix (50:50 ratio of spinach to
pumpkin). The vegetable mix and beef samples were first chopped into small pieces while the
rice was coarsely ground into powder. Then, each sample was homogenized in boiling,
distilled water (1:10 dilution) and extracted to yield at least 10 % (w/w) suspension (Geornaras
et al., 2007). The suspension was homogenized using a kitchen blender for 3 min and then
passed twice through a single layer of cheese cloth to get crude extracts of the food samples.
Nutrient broth was also prepared. Ground samples (0, 10 1nd 30 % ; i.e., 0.0, 2.5 and 7.5 g in
250 ml of extracts) of Tetrapleura tetrapetra, Monodora myristica, Monodora tenuifolia and
Ocimum viride were separately mixed with 250 ml of each of the food extracts or nutrient
broth to give 0.0 %, 1.0 % and 3.0 % of spice per ml of food extracts or broth. These
concentrations of the spices were based on their minimum inhibitory concentrations (MICs)
against the test organisms (E. coli, S. typhimurium and S. aureus) previously determined. They
were autoclaved at 120 OC for 40 min and cooled to about 45 - 48 OC before aseptically
dispersing 65 ml of each into three separate 100 ml sterile amber bottles. The 65ml food
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extracts and broth were cooled to ambient temperature (26 ± 2 OC) and made ready for
bacteriaol inoculation.
3.10.3 Bacterial strains and preparation of inoculants
Inoculants of E. coli 0157, H7, S. typhimurium and S. aureus comprised a mixture of strains
from each pathogen. The strains were sourced from the Department of Microbiology and
virology, Faculty of Medicine, Enugu Campus, University of Nigeria, Nsukka. The strains
were activated and sub-cultured as described by Geonaras and Sofos (2005). From overnight
agar cultures of the test organisms maintained at 37 OC, each of the organisms was adjusted to
105 CFU / ml using Mcfarland-Nephlometer standard (NCCLS, 2006). An aliquote (0.1 ml) of
the adjusted organisms was serially diluted to 106 in 9.9 ml phosphate buffered saline (PBS,
PH7.4; 0.9 g KHPO4.7H20, 8.0 g NaCl and 0.2 g KCl in 1L distilled water), and 1.0 ml
diluents suspended in the sterile 65 ml food extracts and nutrient broth. These were stored at
refrigerated temperature (4 OC) for 10 days for antimicrobial screening.
3.10.4 Monitoring of survival and growth of pathogen population
Survival and growth of the pathogens in the substrates was monitored during storage at 4 OC
for 10 days. This storage temperature permitted normal growth of the pathogens tested in this
study. It is possible to evaluate the antimicrobial activity of spice extracts during simulated,
potential mild temperature abuse of food during distribution, retail or home storage. The
inoculated substrates were not agitated during storage. Surviving populations were enumerated
after incubation on days 2, 4, 7 and 10 of storage. On each of the analysis day, substrates were
thoroughly mixed, and 4ml aliquot of each aseptically removed. These aliquots were serially
diluted in 0.1 % buffered peptone water (Difco), and 1.0 ml of appropriate dilutions (10-6) of
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each of the pathogens, namely E. coli, S. typhii and S. aureus were surface-plated on nutrient
agar. Colonies were manually counted periodically after 24 h of incubation at 37 OC within the
10d refrigerated storage. Detection limit of pathogens was 0.0 CFU / ml.
3.11 Determination of Thiobarbituric acid (TBA) reactive substances in minced meat
patties during storage
Thiobarbituric acid reacts with malonaldehydes to form a pink chromagen, a diadduct of
thiobarbituric acid (TBA) reactive substances (TBARS), which can be detected
spectrophtometrically at 532 nm (Van der Sluis et al., 2000). TBARS was determined as
described by Buege and Aust (1978). On the 1st, 2nd, 4th,7th and 14th days of storage, 5 g from
each of the meat samples was mixed with 2.5 ml of the stock TBA solution {containing 0.375
% TBA (Sigma Chemical Co., St. Louis Mo, U.S.A.) and 15 % Trichloro-acetic acid (TCA)
(Mallinkrodt Beker Inc., Paris ky, USA) in 0.25 N HCL}. The mixture was heated for 10min in
a boiling water bath (100 OC) to develop a pink colour, cooled in tap water and centrifuged
(Beckman coulter Ltd Palo, Alto, Califonia, U.S.A) at 3000 rpm for 20 min. The absorbance of
the supernatants was measured spectrophotometrically (Spectronic 21d, Multon Roy,
Rochester Ny, U.S.A) at 532 nm against a blank that contained all the reagents except the meat
supernatant and multiplied by 2.7 to give thiobarbituric acid (TBA) value in milli
Malonaldehyde equivalent/g.
The percentage inhibition of lipid peroxidation by the spice extracts, expressed as the
inhibition of lipid peroxidation of that sample compared with the lipid peroxidatioon in a
blank, was calculated from the absorbance readings with the expression:
% Inhibition = {(Ablank – Asample) / Ablank} x 100.
Where Ablank = absorbance of the blank, and Asample = absorbance of the sample.
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3.12 Experimental design
Randomized complete block design (RCBD) experiment (Adesoye, 2004) was used in
determining the phenolic (phenol, tannin, flavonoid, β-carotene and anthocyanin) and non-
phenolic phytochemical (phytate, alkaloids, oxalate and saponin) contents in three replicates in
the spices (Tetrapleura tetrapetra, Ocimum viride, Monodora myristica and Monodora
tenuifolia) and spice-treated food extracts. Split-split block design (RCBD) experiments were
conducted to determine antioxidant activities of the 4 spices, using 4 different approaches
(DPPH radical scavenging, TBA test, reducing power and ferric thiocyanate methods); and
antimicrobial properties of the 4 spices at 4 concentrations on 3 bacteria (E. coli, S.
typhimurium and S. aureus) in 4 different food media (micro media, beef, milk and vegetable
mix) for mean (CFU / ml) bacteria populations.
3.14 Statistical analysis
Data generated from all analysis were subjected to analysis of variance and means where
significant (p ≤ 0.05) were separated with Fisher’s least significant difference using Statistical
Package for Social Sciences (SPSS) version 13.0.
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CHAPTER FOUR
4.0 RESULTS AND DISCUSSIONS
4.1 Proximate composition and energy value of spices
Proximate compositions of dried leaves of Ocimum viride, dried seeds of Monodora myristica
and Monodora tenuifolia, and dried fruits of Tetrapleura tetrapetra are shown in Table 6.
Moisture contents of the spices were 9.82 % in O. viride, 9.61% in T. tetrapetra, 8.96 % in M.
tenuifolia and 8.68 % in M. myristica. The values showed that the four spices were relatively
dry (moisture contents less than 12 %) and would store for a long period of time without undue
microbial and biochemical spoilage. Moisture content of any food can be used as an index of
its keeping quality. Water is an important medium for most biochemical reactions. Food
samples with water content of 12 % or more are more prone to high biochemical activities and
usually have short shelf life (Joslyn, 1970).
Crude protein content was highest in M. myristica (22.77%) and was followed by M. tenuifolia
(21.65 %), O. viride (17.85%) and then T. tetrapetra (6.79 %). Thus, M. myristica, M.
tenuifolia and O. viride could serve as excellent sources of protein in the diet if consumed
regularly. Protein is the building block and essential structural component of cells. It provides
the body’s required essential amino acids (Shills and Young, 1988). Protein content in food
varies widely. The spices were also good sources of fat. Fat content was significantly (p <
0.05) lowest (3.44 %) in T. tetrapetra but highest (8.66 %) in M. tenuifolia. M. myristica had
6.34 % and was followed by O. viride which had 4.43 % fat .High fat content implies high
calorific value and possible presence of fat-soluble vitamins, namely vitamins A, D, E and K.
80
Spices are known to be rich sources of essential oils which account for the peculiar aroma
characteristics of the spices (Madsen and Grypa, 2000).
Fibre ranged from 5.25 % in M. myristica to 13.84 % in O. viride but was observed to be
relatively high in T. tetrapetra (29.73 %). Monodora myristica had relatively low fibre content
(5.25 %) compared to 13.8 % content in O. viride and 29.73 % in T. tetrapetra. The high crude
fibre content in T. tetrapetra is not fully exploited because only hot water extract of the fruit is
utilized as spice in most African dishes unlike M. myristica, M. tenuifolia and O viride that are
prepared and consumed alongside the main food items as meals. They could therefore be good
sources of dietary fibre. Dietary fibres are generally plant polysaccharides that cannot be
digested by human digestive enzymes. Dietary fibres are either soluble or insoluble, both
modulate physiological functioning and prevent some degenerative diseases in human. Fibre
in the diet causes variations in the faecal water content, faecal bulk, transit time and
elimination of bile acids and neutral sterols; which lowers the body’s cholesterol pool. Fibres
have been shown to reduce the incidence of coronary and breast cancer (Lintas, 1992; Effiong
et al., 2005).
The spices showed high ash contents (4.17 – 11.75 %). The ash contents of M. tenuifolia
(11.75 %), O. viride (12.44 %) and M. myristica (8.61 %) were significantly higher (p < 0.01)
than that of T. tetrapetra (4.17 %). Ash content refers to the inorganic residues remaining after
either ignition or complete oxidation of organic matter in the sample, and gives an overview of
mineral content of the material (Joslyn, 1970). High ash content implies high mineral contents
in the spices. Monodora tenuifolia, M. myristica and O. viride are likely to be good sources of
minerals in the diet. Nutritionally, ash aids in the metabolism of protein, carbohydrate and fat
(Okaka, 2005)
81
Carbohydrate content ranged from 41.6 % to 47.8 % in the spices.There was no significant
difference (p > 0.05) in carbohydrate contents of the spices. Carbohydrate provides energy to
cells in the body, particularly the brain, the only carbohydrate dependent organ in the body
(Effiong et al., 2005). These spices could be supplementary for carbohydrate need in the diet.
The spices showed high energy values which ranged from 3.11 Kcal in T. tetrapetra to 3.53
Kcal in O. viride. The high energy values of the spices may be of little or no practical
importance in real life situation since the quantity of these spices used in menu is relatively
very small. These spices are consumed in very small amount as food ingredients, and their
contribution to nutrition in menu may not be as high as is the case with staple food items.
However, among many rural consumers who use these spices copiously in various local dishes,
the spices can make meaningful nutritional contribution in menu. Also the high carbohydrate,
protein and energy values of T. tetrapetra may not impart meaningfully to the nutrition of the
users because only the hot water extract of the fruits of this spice is utilized. Generally, the
spices contribute nutritionally to menu and impart many health benefits.
82
Table 6: Proximate composition and energy value of Monodora tenuifolia, Tetrapleura
tetrapetra, Monodora myristica and Ocimum viride
Spices Protein (%) Fat (%)
Ash (%)
Water (%)
Fibre (%)
Carbo-hydrate (%)
Dry matter (%/)
Energy (kJ / kg)
M.
myristica 22.77a 6.34b 8.61b 8.68b 5.25d 46.9a 91.33a 26.62a
M.
tenuifolia 21.65b 8.66a 11.75a 8.96b 7.35c 41.6a 91.07b 18.74b
O. viridi 17.85c 4.43c 12.44a 9.82a 13.84b 42.4a 90.18c 18.93b
T.
tetrapetra 6.79d 3.44d 4.17c 9.61a 29.73a 47.81a 90.11c 16.88b
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05).
83
4.2 Mineral composition of the spices
Table 7 shows the mineral compostion of the four spices. The spices had high contents of most
of the minerals investigated in this study. The most abundant mineral in the spices was iron
which ranged from 10.70 mg / 100 mg in T. tetrapetra, 27.20 mg/100g in M. tenuifolia, and
31.83 mg / 100g in M. myristica to 68.27 mg / 100 g in O. viride. Iron is a major component of
hemoglobin which transports the respiratory gases, namely oxygen (O2) and carbondioxide
(Co2) (Schauss, 1995). Deficiency of iron in the blood stream may lead to death.
Recommended dietary allowance (RDA) for iron is 15mg per day (Food and Nutrition Board,
2001).
The four spices were also good sources of manganese. Manganese content was approximately
12.0 mg / 100 g in both M. myristica and M. tenuifolia, and 16.75 mg / 100 g in O.viride. It
was significantly (p < 0.05) low in T. tetrepetra which had 4.72 mg / 100 g.
Zinc content was high in all the four spices. Zinc ranged from 6.42 to 8.85mg/100g in M.
myristica, M. tenuifolia and O. viride but was relatively low (2.26 mg / 100 g) in T. tetrapetra.
The body contains only a small quantity of biologically active pool of zinc. Therefore, dietary
supply of zinc is continually needed (Schauss, 1995). Zinc is involved in ribonucleic acid
(RNA) and deoxyribonucleic acid (DNA) synthesis needed for cell division, repair and growth
(Food and Nutrition Board, 2001). Zinc may help to prevent growth of abnormal cells
associated with cancer. Zinc as food supplement has been used to enhance wound healing and
improve impaired acuity of taste, smell and night vision. Lack of zinc in the body causes rapid
egestion on the surface of wound and may delay quick healing.
The spices O. viride and M. myristica were significantly (p < 0.05) higher sources of calcium
than M. tenuifolia which also was comparatively a higher source than T. tetrapetra. Ocimum
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viride had about twice (9.67 mg / 100 g) the amount of calcium content (4.84 mg / 100 g) in M.
myristica. The Ca (4.84 mg / 100 g) content in M. myristica was about two and half times the
amount (1.81 mg / 100 g) in M. tenuifolia. Thus, T. tetrapetra is relatively a poor source of
calcium in the diet. Calcium is the most important and most common mineral needed in the
body. Calcium is needed for regulating most internal organs, including the heart and liver. It is
needed for most physiological functional integrity, involving normal functioning of heart
muscles, the skeletal system and cell membrane, blood clotting, nerve signal transmission and
regulation of enzymes and hormones (Food and Nutrition Board, 2001). Deficiency of Ca in
the body leads to malfunctioning of organ systems.
Next in the hierarchy of mineral contents in the spices was magnesium. Ocimum viride had the
highest amount of magnesium (4.40 mg / 100 g), followed by M. tenuifolia (3.27 mg / 100 g),
M. myristica (2.67 mg / 100 g) and finally by T. tetrapetra (2.03 mg / 100 g). Magnesium is
needed for normal functioning of the body. It activates the enzymes necessary for carbohydrate
metabolism (Merki and Merki, 1987; Food and Nutrition Board, 2001).
Sodium contents (0.13 mg / 100 g in M. myristica and M. tenuifolia to 0.99 mg/100 g in O.
viride) were comparatively lower than potassium contents (1.17 in M. myristica to 6.88 mg /
100 g in T. tetrepetra) in any of the four spices. Such low sodium content in relation to
potassium is ideal for normal cell functioning. Sodium and potassium regulate water balance,
heart rhythm, muscles contraction and nerve-signal conduction. Sodium/potassium ratio less
than one (1) is recommended in the body to regulate normal body pH for muscle movement
and nerve irritability. Such balanced sodium/potassium ratio controls glucose absorption and
enhances normal retention of protein during growth (NRC, 1989). It also influences glucose
and lipid metabolism. Increase intake of potassium can lower blood pressure and evidence
85
indicates that it may help prevent strokes. However, extremely high sodium intake has been
associated with fluid retention, leading to hypertension, heart failure and instant death (Talwari
et al., 1989; Food and Nutrition Board, 2001).
Phosphorus, the only nonmetallic micromineral analysed for in this study, occured from 2.00
mg / 100 g to 4.00 mg / 100 g in M. myristica, O. viride and M. tenuifolia. Phosphorus was
relatively low in T. tetrapetra which had as low as 0.86 mg / 100 g of it. Phosphorus is needed
in the diet for good nervous system, strong bone and teeth formation.
All the four spices were poor in selenium content which ranged from approximately 0.4 mg /
100 g to 0.6 mg / 100 g in the spices. While only M. myristica had about 0.4 mg / 100 g, the
other three spces had approximately 0.6 mg / 100 g. Selenium is, however, a micromineral
needed in a very small amount in the body but must be supplied regularly from the diet
Minerals are known to play important metabolic and physiologic roles in living cells (Enechi
and Odonwodo, 2003). Minerals are divided into macro and micro minerals. The
macrominerals, calcium (Ca), magnesium (Mg), sodium (Na) and potassium (K) are required
in large quantities. They are needed in diet in amounts of 100 mg or more per day while the
microminerals iron (Fe), zinc (Zn), selenium (Se) and copper (Cu) are required in less than
100mg per day. Macromineral are required in more than 50 mg per kg of the body weight
while less than 50 mg per kg of the body weight is required of micromineral. It is known that
iron, selenium, zinc, magnessium and manganese strengthen the immune system as
antioxidants (Talwari et al., 1989). Also magnesium, zinc and selenium are known to prevent
cadiomyopathy, muscle degeneration, growth retardation, alopecia, dermatitis, immunologic
disfunctioning, gonadial athrophy, impaired spermatogenesis, congenital malfunctioning and
86
bleading disorder (Chaturvedi et al., 2004). Minerals play important metabolic and
physiological functions in living cells (Enechi and Odonwodo, 2003).
87
Table 7: Mineral composition (mg / 100g) of spices
Spices Ca Fe Mg Mn Zn Na K P Se
M. myristica 4.84b+0.02 31.83b+0.31 2.67c+0.25 11.57c+0.03 6.42c+0.04 0.13c+0.01 1.17d+0.01 2.02c+0.05 0.37c+0.01
M. tenuifolia 1.81c+0.03 27.20c+0.36 3.27b+0.20 11.91b+0.02 7.17b+0.02 0.13c+0.00 2.80c+0.00 4.34a+0.03 0.56a+0.01
O. viridi 9.67a+0.02 68.27a+0.51 4.40a+0.26 16.75a+0.03 8.85a+0.02 0.99a+0.01 8.05a+0.01 3.31b+0.03 0.54b+0.01
T. tetrapetra 0.19d+0.02 10.70d+0.17 2.03d+0.17 4.72d+0..04 2.26d+0.03 0.39b+0.02 6.88b+0.01 0.86d+0.02 0.57a+0.00
LSD 0.042 0.43 0.46 0.03 0.03 0.01 0.01 0.01 0.01
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05).
88
4.3 Vitamin content of the spices
Vitamin contents of the spices are as shown in Table 8. Thiamin, riboflavin and ascorbic acid
(vitamin C) contents were relatively low and ranged from 0.05 mg / 100 g in T. tetrapetra to 0.16
mg / 100 g in M. tenuifolia. Folic acid occurred at the highest level compared to other vitamins in
the spices; it was highest in O. viride (87.51 mg / 100 g) and lowest in T. tetrapetra (13.60 mg /
100 g). It was about three times or more in M. myristica (38.73 mg / 100 g) and M. tenuifolia
(46.10 mg / 100 g) as it was in T. tetrapetra. Niacin content was highest in O. viride (5.47 mg /
100 g), followed by M. tenuifolia (3.63 mg / 100 g), M. myristica (2.95 mg / 100 g) and then T.
tetrapetra (1.25 mg / 100 g). Vitamin C content ranged from 0.34 mg in T. tetrapetra to 0.74 mg
in M. myristica. Vitamin contents of the spices were generally low when compared to vitamin
contents of some commonly consumed green leafy vegetables and fruits which are established
dietary sources of vitamins (Oboh et al., 2006). For example, vitamin C content usually ranged
from 43.5 to 148.0 mg / 100 in green leafy vegetables and from 20 to 29 mg / 100 g in fruits
(Oboh et al 2006).
The likely basis for low vitamin contents in these spices could be attributed to heating effect
during drying of the spices. Sun drying, at least, had been reported to cause a marked decrease in
vitamin contents of food materials. Also, it is evident fron this study that of the four spices, T.
tetrapetra had the least content of each of the vitamins, the least content of crude fat (Table 6) and
the least content of most macrominerals (Table 7) investigated. This low nutrient content could be
the main reason why the natives, even without any scientific knowledge but with long experience
in food and nutrition use only the hot water extract of T. tetrapetra in their diet.
89
Vitamins are generally needed daily in small amounts from foods. They yield no energy directly
but may contribute to energy yielding chemical reactions in the body and promote growth and
development (Murray, 1998). Thiamin, riboflavin, and niacin play key roles as co-enzymes in
energy yielding processes. The recommended dietary allowance (RDA), that is adequate intake, is
1.1 to 1.2 mg for thiamin, 1.1 to 1.3 mg for riboflavin, and 14 to 16 mg for niacin. Only niacin has
upper limit of toxicitv at 35mg or more. They help metabolize carbohydrates, fats and oils.
Enriched grain products are common sources of these three vitamins. Deficiency of the three
vitamins may result in brain damage, poor nervous coordination and disorder in the skins and
gastro-intestinal (GI) tracts of affected persons (Schauss, 1995; Enechi and Odonwodo, 2003).
Folate plays an important role in DNA synthesis and homocysteine metabolism. The RDA for
folate is 400 mg. Excess folate in the diet can mask vitamin B-12 deficiency. Good food sources
of folate include leafy vegetables, organ meat and citrus juices. Its deficiency could lead to
generally poor cell division in various areas of the body, megalloblastic anaemia, tongue
inflammation, diorrhea and poor growth.
Vitamin C is mainly used for synthesizing collagen, a major protein for building connective
tissues. It is a general antioxidant, enhances iron absorption, and is needed for synthesizing some
hormones and neurotransmitters (Food and Nutrition Board, 2001). Vitamin C maintains blood
vessel flexibility and improves circulation in the arteries of smokers. It also acts as an antioxidant
in the body system where it scavenges oxygen-free radicals which are bye-products of many of the
normal metabolic processes in the body (Murray, 1998). Vitamin C deficiency results in scurvy,
which is evidenced in poor wound healing, pinpoint hemorrhages in the skin, and bleeding of gum.
90
A great amount of vitamin C is lost during cooking, and as a result fresh or lightly cooked
vegetables should be included in the diet
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Table 8: Vitamin contents of spices
Spices
Thiamin
(mg/100g)
Riboflavin
(mg/100g)
Niacin
(mg/100g)
Folic acid
(mg /100g)
Ascorbic acid
(mg / 100g)
M. myristica 0.13c+0.01 0.06c+0.00 2.95c+0.03 38.73c+0.21 0.74a+0.00
0.54a+0.01
0.39a+0.01
0.34a+0.00
M. tenuifolia 0.16b+0.01 0.11b+0.00 3.63b+0.02 46.10b+0.61
O. viridi 0.36a+0.00 0.21a+0.01 5.47a+0.03 87.51a+0.99
T. tetrapetra 0.05d+0.00 0.03c+0.00 1.25d+0.01 13.60d+0.06
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05).
92
4.4. Yield of crude extracts of spices
Table 9 shows yields of crude extracts from spices as affected by different extracting solvents.
Very small amounts (0.32 - 0.96 %) of the spices were extracted by the solvents. Yields (%) varied
widely among spices and also among different solvent extracts of the same spices. Methanol (95
%) maintained highest yields of 0.96 % with M. myristica and T. tetrpleura. Also methanol in
combination with hexane and acetone maintained relatively good yields (0.52 – 0.88 %) of
extracts among spices. Water and acetone/water/acetic acid solvents maintained close range of
yields among the spices. Yields of extracts with water were relatively low (0.32 – 0.68 %)
compared to yields of extracts (0.73 – 0.80 %) with acetone/water/acetyic acid.
The solvents are all food grade solvents generally recorgnised as safe (GRAS). Similar solvents
including absolute water, aqueous mixture of ethanol, methanol, hexane and acetone, absolute
methanol, 80 % methanol, 70 % methanol, 95 % ethanol, 80 % ethanol, 70 % ethanol, 80 %
acetone, 70 % acetone and 50 % acetone have been used to extract antioxidants from fruits,
vegetables, legumes and cereals (Shahidi and Naczck, 2004; Sun and Ho, 2005; Xu and Chang,
2007).
Yields of extracts have not always matched propotionally with antioxidant and antimicrobial
activities of the extracts.This is because yields of extracts and composition of yields corelate
independently on the types of solvents with varying polarities and pH, extraction time and
temperature. Under the same condition of extraction, time and temperature, the solvent used and
chemical properties of the food samples ramain two most important factors (Xu et al., 2007, Xu
and Chang, 2007). Thus, high yields of extracts may not always imply high phytochemical
content, antioxidant and antioxidant activities of extracts.
93
Table 9: Yield (%) of crude extracts of spices as affected by different extracting solvents Spices Yields (%) of spice extracts as affected by different extracting solvents
ER OL AW ONE AN MEAN
Monodora
myristica
0.32 0.96 0.80 0.49 0.88 0.69
Monodora
tenuifolia
0.32 0.56 0.80 0.60 0.56 0.57
Ocimum
vinde
0.68 0.32 0.76 0.76 0.52 0.61
Tetrapleura
tetrapetra
0.32 0.96 0.73 0.32 0.60 0.58
ER = distilled water, OL = 95% methanol, ONE = acetone/hexane (1:1; v/v), AN = hexane/methanol/acetone (2:1:1; v/v/v/v), AW = acetone/water/acetic acid (70:29.5:0.5; v/v/v)
94
4.5 Effects of different extraction solvents on non-phenolic phytochemical contents of the
spices
4.5.1 Alkaloid
Alkaloid content of five different solvent extracts from four Nigerian spices is presented in Table
10. Spice extracts from different extraction solvents differed significantly (p < 0.05) in their
alkaloid content.The alkaloid content of Monodora myristica from different extraction solvents
ranged from 2.07 to 5.76 mg / 100 g, Monodora tenuifolia from 0.80 to 6.54 mg / 100 g, Ocimum
viride from 3.64 to 5.42 mg / 100 g, and Tetrapleura tetrapetra from 2.85 to 5.08 mg / 100g.
Generally, alkaloid content of the four spices were significantly (p < 0.05) affected by the different
solvents used. The alkaloid contents of the spices as affected by the extracting solvents were in the
following order from high to low: acetone / hexane (1:1; v/v) >acetone / water / acetic acid
(70:29.5:0.5; v/v/v) > hexane / methanol / acetone (2:1:1; v/v/v) > 95 % methanol > distilled water
for monodora myristica; acetone / water / acetic acid > acetone / hexane > 95 % methanol >
hexane / methanol / acetone > distilled water for Monodora tenuifolia; hexane / methanol /
acetone > distilled water > acetone / hexane > 95 % methanol, and acetone / water / acetic acid for
Ocimum viride; and acetone / water / acetic acid (70:29.5:0.5; v/v/v) > acetone / hexane > 95 %
methanol > hexane / methanol / acetone > distilled water for Tretapleura tetrapetra. These results
suggest that types of spices being extracted and types of solvent used influence the quantity of
alkaloid extracted. It was also evident that the solvents work better when in combination than
when used singley for alkaloid extraction from the spices. Distilled water was the weakest
95
extraction solvent while acetone in combination with the other solvents, including hexane,
methanol, acetic acid and distilled water was the best extraction solvent.
The presence of alkaloids in plants such as Moringa oleifera elevated the plant to such an
important position to treat hypertension. Many plants containing alkaloids and flavonoids have
diuretic, antispasmodic, anti-inflammatory and analgesic effect (Owoyele et al., 2002; Ujowundu
et al., 2010). The high alkaloid content of these spices could account for their popular use in the
traditional treatment of hypertension. These secondary metabolites have been associated with
numerous physiological activities in mammalian cells in various studies (Sofowora, 1993; Abo et
al., 1999; Nweze et al., 2004; Mishra et al., 2009).
96
Table 10: Effect of different extraction solvents on alkaloid contents (mg / 100 g) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Monodora
myristica
2.07c ± 0.02 4.88b ±0.04 5.22ab ± 0.06 5.76a ± 0.02 5.04b ±0.01 4.59±0.61
Monodora
tenuifolia
0.8c ± 0.01 3.66c ± 0.04 6.54a ± 0.03 4.54b ±0.05 3.05d ±0.02 3.72±1.81
Ocimum
vinde
5.08a ± 0.03 3.64c ± 0.02 3.73c ±0.02 4.67b ±0.02 5.42a ± 0.01 4.51±0.06
Tetrapleura
tetrapetra
2.85c ± 0.01 3.96b ±0.02 5.08a ± 0.01 4.32b ±0.01 3.25S ± 0.08 3.89±0.32
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95% methanol, ONE = acetone/hexane (1:1; v/v), AN = hexane/methanol/acetone (2:1:1; v/v/v/v), AW = acetone/water/acetic acid (70:29.5:0.5; v/v/v)
97
4.5.2 Oxalate
In order to estimate the potential of oxalate as a bioactive ingredient of selected spices in foods
and other biological materials, oxalate content of various solvent extracts of the spices was
analysed, and the results presented in Table 11. Different extraction solvents of the spices differed
significantly (p < 0.05) in their oxalate contents. The oxalate content of M. myristica ranged from
2.0 to 3.5 mg / 100 g; M. tenuifolia from 3.5 to 7.0 mg / 100 g; O. virdie from 3.0 to 4.0 mg / 100
g, and T. tetrapetra from 3.5 to 5.6 mg / 100 g. The oxalate content yields by the extraction
solvents were in the following order from high to low: Acetone/hexane > acetone / water / acetic
acid > Distilled water, and hexane / methanol / acetone > 95 % methanol for M. myristica;
distilled water > acetone/water/acetic acid > acetone/hexane and hexane/methanol/acetone > 95 %
methanol for M. myristica; Acetone/water/acetic acid > 95 % methanol, and hexane / methanol
/acetone > distilled water, and acetone/hexane for O.viride; and distilled water > hexane /
methanol / acetone > 95 % methanol > Acetone / water / acetic acid, and acetone/hexane for T.
tetrapetra. These results suggest that distilled water was the best among the five extraction
solvents for extracting oxalate from M. tenuifolia and T. tetrapetra while acetone / water / acetic
acid was the best solvent for O.viride, and acetone/hexane was the best for M. myristica. Thus,
distilled water or distilled water in combination with other solvents seemed to be the best for
extracting oxalate from spices. Also M. tenuifolia was highest in oxalate content, followed by T.
tetrapetra, O. viride and then M. myristica.
The oxalic acid content of vegetables has been used as an index of their toxicity since a high
content of it would lower the nutritive value of food (Ujowundu et al., 2010). Oxalic acid in plants
contributes to antioxidant properties and hence the therapeutic potentials of the spices. Oxalic acid
98
content in food would be an index of toxicity level of the food. However, oxalate at low level
advantageously confers antioxidant activity in both food and human. Dietary oxalate has also been
shown to complex with calcium, magnesium and iron, forming insoluble oxalate salts which cause
oxalate stone (Oke, 1966). Oxalic acid chelate radical-initiating divalent metals thereby reducing
incidence of oxidative degenerative diseases in human.
99
Table 11: Effect of different extraction solvents on oxalate contents (mg / 100 g) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Monodora
myristica
2.5c ±0.01 2.0d ±0.02 3.0b ± 0.01 3.5a ± 0.00 2.5c ± 0.00 2.7±0.06
Monodora
tenuifolia
7.0a ±0.04 3.5c ± 0.00 4.0b ± 0.01 4.0b ± 0.01 4.0b ± 0.01 4.5±0.61
Ocimum
vinde
3.0c ± 0.01 3.5b ± 0.01 4.0a ± 0.02 3.0c ± 0.10 3.5b ± 0.02 3.6±0.08
Tetrapleura
tetrapetra
5.55a ±0.04 4.0b ± 0.02 3.5c± 0.01 3.5c ± 0.02 4.5ab ±0.03 4.21±0.32
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95% methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
100
4.5.3 Saponin
The effects of various solvent extraction systems on recovery of saponin from the selected spices
are presented in Table 12. Spice extracts from different extraction solvents differed significantly (p
< 0.05) in their saponin content. Saponin contents of M. myristica ranged from 0.01 to 0.74 mg /
100 g; M. tenuifolia from 0.01 to 0.29 mg / 100 g; O. viride from 0.14 to 0.62 mg / 100 g; and T.
tetrapetra from 0.16 to 0.60 mg/100 g. Generally, saponin content in the four spices was relatively
low when compared with the compositions of other non-phenolic phytochemicals. The saponin
yields by the extracting solvents were in the following order from high to low: 95 % methanol >
acetone/hexane (1:1; v/v) > hexane / methanol / acetone (2:1:1; v/v/v) > acetone / water / acetic
acid (70:29.5:0.5; v/v/v) > distilled water for M. myristica; acetone / water / acetic acid > acetone /
hexane > distilled water > 95 % methanol > hexane / methanol / acetone for M. tenuifolia; hexane
/ methanol / acetone > acetone / water / acetic acid > acetone / hexane > 95 % methanol >
distilled water for O.viride; and distilled water > 95 % methanol > acetone / water / acetic acid,
and acetone/water > hexane / methanol / acetone for T. tetrapetra. The results showed that
saponin contents of the spices was low and differed (p < 0.05) significantly.among the spices. The
spice T. tetrapetra had the highest saponin content among the four spices.
Saponins possess carbohydrate moieties attached to tetraprenoid or steroidal aglycones (Sridhar
and Bhat, 2007). Saponins constitute a key ingredient in traditional Chinese medicine and are
responsible for many of the attributed biological effects. They reduce uptake of glucose and
cholesterol through intra-lumenal physicochemical interaction during food transition in the gut.
This could confer chemo-protection against heart diseases.
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Table 12: Effect of different extraction solvents on saponin contents (mg / 100 g ) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Monodora
myristica
0.016d ± 0.00 0.74c ± 0.02 0.50c ± 0.01 0.72a ± 0.01 0.60b ±0.01 0.41±0.13
Monodora
tenuifolia
0.16bc ± 0.00 0.10c ± 0.00 0.29a ± 0.01 0.23a ± 0.00 0.06c ± 0.00 0.17±0.08
Ocimum
vinde
0.14b ± 0.00 0.19b ± 0.01 0.4a ± 0.02 0.38a ± 0.02 0.06c ± 0.05 0.15±0.04
Tetrapleura
tetrapetra
0.60a ± 0.02 0.44b ± 0.01 0.33c ± 0.01 0.03e ± 0.01 0.16d ±0.02 0.31±0.09
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.5.4 Phytate
Phytate contents of solvent extracts of the four spices are presented in Table 13. The phytate
contents of the different solvent extracts ranged from 2.14 to 2.38 mg / 100 g in M. myristica;
from 3.02 to 5.50 mg / 100 g in M. tenuifolia; from 2.32 to 2.62 mg / 100 g in O.viride; and from
3.24 to 3.80 mg / 100 g in T. tetrapetra. Spice extracts from different extraction solvents differed
significantly (p < 0.05) in phytate contents. Mean while, each spice had a close range of values of
phytate content among its different extraction solvents. The phytate content of the different
extraction solvents were in the following order from high to low: acetone / water / acetic acid
(70:29.5:0.5; v/v/v), and acetone / hexane (1:1; v/v) > hexane / methanol / acetone (2:1:1; v/v/v) >
distilled water, and 95 % methanol for M. myristica; distilled water > acetone / water / acetic acid ,
acetone / hexane and hexane / methanol / acetone > 95 % methanol for M. tenuifolia; distilled
water, 95 % methanol and acetone / water / acetic acid > acetone / hexane, and
hexane/methanol/acetone for O. viride; and acetone / hexane > hexane / methanol / acetone >
distilled water > 95 % methanol > acetone / water / acetic acid for T. tetrapetra. These results
suggest that the extractability of phytate by the extracting solvents varied with the type of spice
being extracted; and that distilled water was the best extracting solvent for M. tenuifolia and O.
viride while acetone/hexane was the best for T. tetrapetra. The spices could serve as good sources
of phytate in food and food related systems due to the high content of phytate in them.
Phytate is a natural plant inositol hexaphosphate constituting about 1.5 % of many plants (Reddy
et al., 1989). Phytate is a very stable and potent chelating food component that is considered to be
an antinutrient by virtue of its ability to chelete divalent metals and prevent their absorption
(Oboh, 2006). However, it has also been shown to have anticancer and antioxidant activiity. It
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forms an iron chelate that suppresses lipid oxidation by blocking iron driven hydroxyl radical
generation. Metal phytate complexes are highly insoluble over a wide range of pH and as a result
inhibit iron-related hydroxyl radical formation by forming an inactive iron-chelate (Graf and
Eaton, 1990).
Presence of phytate in foods has been associated with reduced mineral absorption due to the
structure of phytate with high density of negatively charged phosphate groups which can complex
with many mineral ions, causing non-availability for intestinal absorption. However, presence of
phytate in high fibre foods may reduce the incidence of breast cancer and cardiovascular diseases.
Phytates are stable compounds that chelate excess divalent metals and control their excess
absorption, thereby lowering the incidence of cancer in human (Oboh, 2006).
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Table 13: Effect of different extraction solvents on phytate contents (mg / 100 g ) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Monodora
myristica
2.14b ±0.00 2.15b ±0.00 2.37b ± 0.02 3.38a ± 0.02 2.32b ± 0.02 2.27±0.06
Monodora
tenuifolia
5.50a ±0.02 3.02c ±0.00 3.88b ± 0.10 3.84b ±0.04 3.84b ± 0.02 4.02±0.89
Ocimum
vinde
2.62a ±0.02 2.63a ±0.02 2.61a ± 0.01 2.34b ±0.00 2.32b ± 0.04
2.51±0.32
Tetrapleura
tetrapetra
3.38ab ±0.02 3.24bc±0.02 2.79c ± 0.01 3.8a ±0.04 3.43ab ± 0.00 3.33±0.03
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.6 Effects of different extraction solvents on phenolic phytochemical contents
of the spices
4.6.1 Total phenol content, garlic acid equivalent per 100 g (GAE / 100 g)
Table 14 reports total polyphenols contents in different solvent extracts of four Nigerian spices,
namely dried leaves of Ocimum viride, dried fruits of T. tetrapetra, dried seeds of Monodora
myristica and Monodora tenuifolia. The various solvent extraction systems [distilled water, 95 %
methanol, acetone / hexane (1:1; v/v), hexane / methanol / acetone (2:1:1; v/v/v), and acetone /
water / acetic acid (70:29.5:0.5; v/v/v)] affected recovery of total phenols from the selected spices.
Spice extracts from different extraction solvents differed significantly (p < 0.05) in their total
phenol contents (TPCs). The TPCs of O. viride ranged from 8.77 to 12.33 garlic acid equivalent /
100 g; M. myristica from 2.43 to 7.45 GAE / 100 g; M. tenuifolia from 2.61 to 6.78 GAE / 100 g;
and T. tetrapetra from 0.21 to 15.93 GAE / 100 g. The TPCs yields by the extracting solvents
were in the following order from high to low: acetone / hexane > hexane/methanol/acetone > 95 %
methanol > distilled water > acetone / water / acetic acid for Ocimum viride; distilled water >
acetone / hexane > hexane / methanol / acetone > 95 % methanol > acetone water / acetic acid for
Monodora myristica; acetone / hexane >distilled water > hexane / methanol / acetone > 95 %
methanol > acetone / water / acetic acid for Monodora tenuifolia; and 95% ethanol > acetone /
hexane > hexane / methanol / acetone > distilled water > acetone / water / acetic acid for
Tetrapleura tetrapetra.
These results showed the solvent combination acetone / water / acetic acid (70:29.5:0.5, v/v/v) as
the best extractants for total phenol from any of the four spices. Among the four spices analysed,
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Ocimum viride had the highest total phenol content, followed by Tetrapleura tetrapetra.
Generally, the four spices are good sources of phenols.
Phenols are one of the major groups of non-nutritive dietary components that have been associated
with the inhibition of cancer, atheriosclerosis, as well as ameloriating age-related degenerative
brain disorder (Wang et al, 1998; Chang et al., 2002). Phenolic phytochemicals inhibit
autoxidation of unsaturated lipids, thus preventing formation of oxidized low density lipoprotein
(LDL) which has been associated with the incidence of cardiovascular diseases (Fang et al., 2002;
Xu et al., 2007). Natural phenolic compounds are capable of decreasing oxygen concentration,
intercepting singlet oxygen, preventing 1st – chain initiation by scavenging initial radicals such as
hydroxy radicals, binding metal ion catalyst, decomposing primary products of oxidation to
nonradical species, and breaking chains to prevent continued hydrogen abstraction from
substances (Shahidi and Naczk, 2004). Phenolic compounds play important roles in stabilizing
lipid peroxidation and are associated with antioxidant activity (Yen et al., 1993). They may
contribute directly to the antioxidant action (Duh et al., 1999). According to Tanaka et al. (1998),
polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in human
when up to 1.0 mg is ingested daily from diets rich in fruits and vegetables.
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Table 14: Effect of different extraction solvents on phenol contents (GAE / 100 g ) of spices
Species Extracting solvents
ER AW AN ONE OL MEAN
Ocimum
vinde
10.33b ±0.03 16.77a±0.04 7.68c ±0.02 6.18e ±0.01 9.03d ±0.02 11.71±2.06
Monodora
myristica
2.13e ±0.01 7.06a ±0.02 4.82c ±0.01 4.28d± 0.02 6.18b ± 0.02 5.66±1.12
Monodora
tenuifolia
2.40d ±0.00 8.85a ± 0.01 2.63c ± 0.02 2.30d± 0.01 5.50b ±0.02 5.04±1.81
Tetrapleura
tetrapetra
7.17c ±0.13 13.93a ± 0.61 3.14d±0.01 3.04d ± 0.00 10.47b ± 0.00 7.24±1.22
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.6.2: Condensed tannin content
Table 15 shows tannin contents of the four spices. Tannins are produced via condensation of
simple phenolics and exist in different molecular weights. Tannins are generally divided into
hydrolysable and condensed proanthocyanidins (polymers of flavan-5-ol(s) (Hansen et al., 1989).
Spice extracts from different extraction solvents differed significantly (p < 0.05) in their
condensed tannin contents (CTCs). The CTCs of Ocimum viride ranged from 0.07 mgTAE / 100 g
in distilled water extract to 0.20 mg TAE / 100 g in hexane/methanol/acetone extract; Monodora
myristica from 0.02 mg TAE / 100 g in 95 % ethanol extract to 0.15 mg TAE / 100 g in
acetone/water/acetic acid extract; Monodora tenuifolia from 0.01 mg TAE / 100 g in
acetone/hexane extract to 0.09mg TAE/100g in distilled water extract; and Tetrapleura tetrapetra
from 0.02 mg TAE / 100 g in 95 % methanol extract to 0.08 mg TAE / 100 g in
hexane/methanol/acetone extract. These results suggest that acetone/hexane solvent system was
the best for extracting condensed tannin from Ocimum viride, and Tetrapleura tetrapetra;
acetone/water/acetic acid solvent system was the best for Monodora tenuifolia while
acetone/hexane solvent system was the best for Monodora myristica.
The presence of tannins in these spices supports their uses in traditional medicine for the treatment
of different diseases. Shahidi (1996) reviewed biological activities of tannins and noted that
tannins have remarkable activity in cancer prevention and anticancer activities. Edible plant
materials containing tannins are known to be astringent, and are used for treating intestinal
disorders such as diarrhea and dysentery (Smirnoff, 2000).
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Table 15: Effect of different extraction solvents on tannin contents (TAE / 100 g ) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Ocimum
vinde
0.07b ±0.01 0.11b ±0.02 0.11b ± 0.04 0.20a ± 0.02 0.11b ±0.03 0.12±0.02
Monodora
myristica
0.05c ± 0.02 0.15a ± 0.04 0.05c ± 0.02 0.11b ±0.01 0.2d ± 0.00 0.12±0.03
Monodora
tenuifolia
0.09a ± 0.03 0.07ab±0.02 0.03c ± 0.00 0.01c ± 0.01 0.06b ±0.02 0.05±0.02
Tetrapleura
tetrapetra
0.05c ±0.00 0.05c ±0.00 0.08b ±0.03 0.09a± 0.02 0.02d ±0.00 0.06±0.00
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.6.3 Total flavonoid content
Flavonoids are widespread plant secondary matabolites, namely flavones and flavanols. In in vitro
analysis, flavonoids from several plant sources exhibited free radical scavenging activity and
protect against oxidative stress in rat liver microsomes (Xu and Chang, 2007). As components of
fruit, vegetables and spices, flavonoids are regularly contained in human food. However, there are
only few documented reports on identification and quantification of flavonoids in foods (Romani
et al., 2004; Xu and Chang, 2007; Xu et al., 2007).
Total flavonoid contents (TFCs) in Ocimum viride, Monodora myristica, Monodora tenuifolia, and
Tetrapleura tetrapetra extracted with different extraction solvents differed significantly (p < 0.05)
(Table 16). The TFC of Ocimum viride ranged from 0. 22 mg GAE / 100 g to 0.28 mg GAE / 100
g; Monodora myristica from 0.09 to 0.91 mg GAE / 100 g; Monodora tenuifolia from 0.05 to 0.09
mg GAE / 100 g; and Tetraplenra tetrapetra from 0.05 to 0.07 mg GAE / 100 g. These spices
were generally low in flavonoid contents. However, Ocimum viride had the highest while
Tetrapleura tetrapetra had the least total flavonoid content. Within each spice, the total flavonoid
contents of the various solvent systems did not differ much from each other, depicting that the five
solvent systems have almost equal extraction capacity for total flavonoid content. However, from
the result, 95% methanol was the best overall extracting solvent for TFC across the four spices.
Flavonoids in human diet may reduce the risk of various cancers as well as prevent menopausal
symptoms. Epidemiological studies suggest that consumption of flavonoid was effective in
lowering the risk of coronary heart diseases (Rice-Evans et al., 1996). Flavonoids in human diet
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may reduce the risk of various cancers as well as prevent menopausal symptoms (Ross and
Kasum, 2002; Padayatty et al., 2003).
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Table 16: Effect of different extraction solvents on flavonoid contents (GAE / 100g) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Ocimum
vinde
0.23c ± 0.04 0.24b ±0.03 0.28a ± 0.01 0.22d ±0.02 0.28a ± 0.02 0.25±0.00
Monodora
myristica
0.09c ± 0.03 0.09c ±0.02 0.11a ± 0.02 0.10b ± 0.01 0.10b ±0.00 0.10±0.00
Monodora
tenuifolia
0.05c ± 0.01 0.08b ±0.00 0.09a ± 0.01 0.09a ± 0.02 0.08b ±0.02 0.08±0.01
Tetrapleura
tetrapetra
0.05b ±0.02 0.07a ±0.00 0.07a ±0.00 0.07a± 0.00 0.07a ±0.03 0.07±0.00
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.6.4 Total carotenoid content
Spice extracts from different extraction solvents differed significantly (p < 0.05) in total
carotenoid content (TCCs) (Table 17). The four spices, except Monodora myristica and Monodora
tenuifolia exhibited different total carotenoid contents. The TCC ranged from 0.14 to 93mg GAE /
g in O. viride; from 0.03 to 0.28 mg GAE / g in M. myristica; from 0.00 to 0.221mg GAE / g in M.
tenuifolia; and from 0.081 to 0.22 mg GAE / g in T. tetrapeta. Distilled water seemed to be the
best extracting solvent for TCC from M. myristica and M. tenuifolia; acetone/water/acetic acid and
95 % methanol solvent systems were the best for O. viride while hexane/methanol/acetone solvent
system was the best for T. tetrapetra.
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Table 17: Effect of different extraction solvents on carotenoid contents (GAE / 100 g ) of
spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Ocimum
vinde
0.86ab±0.02 0.93a ±0.02 0.14c ± 0.01 0.74b ±0.00 0.93b ±0.03 0.55±0.01
Monodora
myristica
0.28a ±0.02 0.03c ±0.00 0.07b± 0.00 0.00d ±0.00 0.03c ± 0.00 0.08±0.02
Monodora
tenuifolia
0.26a ±0.01 0.00c ±0.00 0.05b ±0.00 0.16a ± 0.01 0.07b ±0.00 0.12±0.01
Tetrapleura
tetrapetra
0.23a ±0.02 0.08b ±0.01 0.26a ± 0.03 0.23a ± 0.04 0.02c ± 0.01 0.08±0.02
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol /acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.6.5 Total anthocyanin content
Table 18 shows anthocyanin contents of the four spices. Anthocyanin was very low in the four
spices, suggesting that these spices are not good dietary sources of anthocvanin. Spice extracts
from different extraction solvent differed significantly (p < 0.05) in their anthocyanin contents.
Anthocyanins are known to inhibit LDL oxidation and LDL-mediated macrophage apoptosis,
serving as a chemo-preventive agent (Tseng et al., 1992). Anthocyanins may be useful in
preventing the deleterious consequences of oxidative stress; and that is the main reason for
increasing concern in the protective biochemical functions of many natural bioactives, including
anthocyanin in spices, herbs and medicinal plants (Osawa and Namiki, 1981).
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Table 18: Effect of different extraction solvents on anthocyanin contents
(mg Cyanidin-3-glucoside / g) of spices
Species Extracting solvents
ER OL AW ONE AN MEAN
Ocimum
vinde
0.05a ±0.02 0.02c ± 0.01 0.01d ± 0.01 0.05a ±0.01 0.03b ± 0.00 0.03±0.00
Monodora
myristica
0.02b ± 0.00 0.01c ±0.00 0.06a ±0.01 0.02b ±0.00 0.02b ± 0.00 0.03±0.01
Monodora
tenuifolia
0.00c ± 0.00 0.02b ±0.00 0.02b ±0.00 0.02b ±0.00 0.03a ± 0.01 0.02±0.00
Tetrapleura
tetrapetra
0.03c ±0.01 0.05a ±0.01 0.04b ±0.02 0.02d ±0.01 0.02d V0.00 0.03±0.01
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same row are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone/hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
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4.7 Estimation of reducing power of spices 4.7.1 Reducing power of Ocimum viride
The reducing power of solvent extracts of the spices Ocimum viride is shown in Fig 1, Monodora
myristica in Fig 2, Mnodora tenuifolia in Fig 3 and Tetrapleura tetrapetra in Figs 4. Each of the
spices was extracted with distilled water, 95 % methanol, acetone/hexane (1:1, v/v),
hexane/methanol/acetone (2:1:1, v/v/v/v) and acetone/water/acetic acid (70:29.5:0.5, v/v/v); and
the reducing power investigated by measuring the conversion of Fe3+ to Fe2+. The reducing
capacity of a compound is a significant indicator of its potential antioxidant activity (Meir et al.,
1995). Antioxidant activities of putative antioxidants are attributed to various mechanisms such as
prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxide,
prevention of continued proton abstraction, and radical scavenging (Diplock, 1997). Huang et al.
(2005) recently reported that a reducing property can be a novel defence mechanism against lipid
peroxidation. This is possible through the ability of the antioxidant compounds to reduce transition
metals such as Fe2+ or Cu+. Reduceed metals rapidly react with lipid hydroperoxide, leading to the
formation of reactive lipid radicals and conversion of the reduced metals to their oxidized forms.
The reducing power of various solvent extracts of Ocimum viride increased with increasing
concentrations of extract in the solvents, and differed significantly (p < 0.05) among different
solvent extracts (Fig. 1). Methanol extract exhibited the highest reducing power among the five
solvent extracts, followed by the hexane/methanol/acetone extract. The reducing power of the
methanol extract ranged from 0.65 to 1.63 nanometer (nm). Water extract had the lowest reducing
power which ranged from 0.20 to 0.26. There was no significant difference (p > 0.05) between the
reducing power of acetone/water/acetic acid and that of acetone/hexane extracts; and their
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reducing power ranged from 0.06 to 0. 56 nm. High value of reducing power of any biological
material indicates high antioxidant activity.
119
Fig. 1. Antioxidant activity of different solvent extracts of Ocimum viride. Values represent means of three determinations of different samples, ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
120
4.7.2 Reducing power of of Monodora myristica
The reducing power of the five solvent extracts of dried seeds of Monodora myristica is shown in
Figure 2. The different solvent extracts exhibited a dose-dependent reducing power within
concentration range of 4, 8, 12, 16 and 20 mg dry matter/ml in the final concentrations. Methanol
extract of the spice had the highest reducing power, followed by acetone/hexane extract,
hexane/methanol/acetone extract, and then water extract. The acetone/water/acetic acid extract had
the least reducing power. The different solvent extracts of the spice showed significant (p < 0.05)
differences in reducing power. The 95 % methanol solvent extract of Monodora myristica showed
highest reducing power and high reducing power implies high antioxidant activity of the
extracts.Thus 95 % methanol is an excellent solvent for extracting natural antioxidant components
of the spice for broader application.
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Fig. 2. Antioxidant activity of different solvent extracts of Monodora myristica. Values represent means of three determinations of different samples, ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v).
122
4.7.3 Reducing power of Monodora tenuifolia
Fig 3 shows the reducing power of various solvent extracts of ground, dry seeds of Monodora
tenuifolia. Reducing power of the extracts increased with increasing concentrations (mg dry matter
/ ml) of the spice in all the solvents and was affected by the characteristics of the solvents used.
Methanol extract had the highest reducing power among the five extraction solvents, with
reducing power ranging from 0.16 to 1.07 nm within the 4, 8, 12, 16 and 20 mg dry matter / ml of
the spice used in this study.
However, reducing power did not differ (p > 0.05) significantly among the remaining four solvent
extract within 2 to 16 mg / ml concentrations of the spice in the solvents but differed significantly
(p < 0.05) at higher 20mg/ml concentration. Hexane/ methanol/acetone extract had the least
reducing power while ditilled water extract exhibited the highest reducing power at 20 mg / ml
concentration. This result showed that M. tenuifolia exhibited reducing ability and that 95 %
methanol was the best extracting solvent for reducing power characteristics of the spice.
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Fig. 3. Antioxidant activity of different solvent extracts of Monodora tenuifolia. Values represent means of three determinations of different samples analysed, ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
124
4.7.4 Reducing power of Tetrapleura tetrapetra
The reducing power of the different solvent extracts of ground, dry fruits of Tetrapleura tetrapetra
is shown in Fig. 4. Reducing power of the solvent extracts increased with increasing
concentrations (mg dry matter / ml) of the spice in the solvents. The reducing power was affected
by the extracting solvents in the following order from low to high: water > 95 % methanol >
hexane / methanol / acetone > acetone / hexane > acetone / water / acetic acid. Thus, T. tetrapetra
possess reducing ability, and water extract exhibited the best reducing power suggesting water as
the best extractant for optimal reducing power.
The redncing power of these spices is a measure of their free radical scavenging, election donating
and sulfhydryl-donating capacity. Reducing power is also the key to the multiple actions of the
spices at the molecular, cellular and tissue levels, and to their effectiveness as a systemic
protectant against oxidation and free radical damages in the cells.
125
Fig . 4. Antioxidant activity of different solvent extracts of Tetrapleura tetrapetra. Values represent means of three determinations of different samples analysed, ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane/methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
126
4.7.5 Comparative effect of extracting solvents on reducing power of the four spices
Fig. 5 compares effects of the five different extracting solvents on reducing power of the four
spice extracts. Higher reducing power implies higher antioxidant activity. Antioxidant activities of
extracts of each spice were significantly (p < 0.05) influenced by the extracting solvents and the
best extracting solvent for one spice was not always for another spice. Water extracts of Ocimum
viride and Monodora myristica exhibited the least reducing power (0.263 and 0.303 respecctively)
among the various solvent extracts of each spice whereas water extract of Monodora tenuifolia
exhibited the highest reducing power (1.59) among the fivee solvent extracts of the spice.
The relative differences in reducing power of various solvent extract of the four spices are
conspicuously exhibited in the bar chart (Fig. 5). Generally, methanol extracts of all the spices
showed relatively high reducing power, with that of Tetrapleura tetrapetra exhibiting the highest
reducing power when compared with methanol extract of the other spices. When this result (Fig.
5) is compared with those of Figs 1 to 4, one would understand that the spice and solvent
characteristics and the concentration of the spices in the solvents all affect the reducing power of
the extracts
Binding of transition metal ion is one of the mechanisms through which antioxidants inhibit lipid
peroxidation in biological and food systems. In this manner antioxidants reduce oxidative state of
transition metal ions from high to low oxidation number, thereby reducing their carcinogenic
effects in human. Plant materials, including spices, with high reducing power are usually good
natural antioxidants. The reducing power of the four spices could be summerised in the following
order from high to low: T. tetrapetra > M. tenuifolia > O. viride > M. myristica.
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Fig. 5. Comparative reducing power of spice extracts from different solvents. ER = distilled water, OL = 95 % methanol, ONE = acetone/ hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
128
4.8 Inhibition of Linoleic acid peroxidation
Figure 6 shows the percentage inhibition of linoleic acid of various solvent extracts of the spices at
20 mg dry matter/ml, using ferric thiocyanate method (FTC). Ferric thiocyanate method estimates
the ability of the antioxidant compounds to suppress pro-oxidant and oxidant activities in
oxidizing systems. It is most appropriate at the initial stage of lipid peroxidation, and in
estimating delay or prevention of low density lipoprotein (LDL) peroxidation by antioxidants.
Delay or prevention of linoleic acid peroxidation is an indication of antioxidant activity. A higher
percentage inhibition indicates higher antioxidant activity.
All the solvent extracts except water extract of M. tenuifolia exhibited more than 50 % linoleic
acid peroxidation. The ranges of peroxidation inhibition (%) were 43.5 for water extract to 89.5
for 95 % methanol extract of Monodora tenuifolia, 72.6 for acetone/water/acetic acid extract to
88.70 for water extract of Tetrapleura tetrapetra, 65.5 for hexane/methanol/acetone extract to 94.6
for water extract of M myristica, and 69.6 for acetone/hexane extract to 94.13 for water extract of
Ocimum viride. Water extracts of Ocimum viride and Tetrapleura tetrapetra, acetone/water/acetic
acid extract of Monodora myristica and 95 % methanol extract of Monodora tenuifolia
significantly (p < 0.05) exhibited highest percentage linoleic acid inhibition for each spice.
129
Fig.6. Inhibition of linoleic acid per oxidation by different solvent extracts of spices. ER = distilled water, OL = 95 % methanol, ONE = acetone/hexane (1:1; v/v), AN = hexane/methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v).
130
4.9.1 Scavenging of 1,1 – diphenyl-2-picryl hydrazyl radical (DPPH) by the spices
The scavenging potentials of various solvent extracts of the four spices against DPPH radical is
shown in Table 19. The five solvent extracts had exceptionally high scavenging activity
suggesting that the spices have good radical scavenging constituents. The extracting solvents
affected radical quenching capacity of the spices. Generally a stronger radical quenching agent has
a lower IC50 value. The water extracts of M. myristica (3.29 mg / g) and T. tetrapetra (3.78 mg /
g), hexane/methanol/acetone extract of M. tenuifolia (3.70 mg / g) and acetone/water/acetic acid
extract of O. viride (3.64 mg / g) had the lowest IC50 among the five solvent extracts of each spice.
Evidently, the IC50 of the four spices, regardless of type of solvent used, fall within a very close
range of 3.29 to 3.78. Water alone and methanol in combination with other solvents were the best
extracting solvents for radical scavenging antioxidant properties.
The 1,1-dyphenyl-2-picryl hydrazyl (DPPH) radical was widely used in model system to
investigate the scavenging activities of several natural compounds such as phenolics and
anthocyanins or crude mixtures such as solvent extracts of plants (Huang et al., 2005).
The antioxidant screening method using 1, 1-diphenyl-2-picry1hydrazyl (DPPH) radical
scavenging activity is based on its reduction and decolourisation of its purple colour to reduced
yellow colour 2,2-diphenyl-1-picry1hdrazine, stable free radical by an electron- or hydrogen-
donating molecule, that is free radical scavenging antioxidant. As the odd electron of the radical
becomes paired off in the presence of a hydrogen donor, that is a free radical scavenging
antioxidant, the absorption strength decreases and this is quantified stiochiometrically with respect
to the number of electrons captured. Because of its odd electron, 1, 1-diphenyl-2-picry1hdrazyl
gives a strong absorption maximum at 527nm by visible spectrophotometer (purple colour). An
131
increase in the amount of antioxidant compounds present in the extracts result to an increase in the
DPPH free radical scavenging activity, and lower absorbance values.
132
Table 19: Free radical 1,1-diphenyl-3-picryl hydrazyl(DPPH) Scavenging activity by
different solvent extracts of spices.
IC50 (mg / ml)
Solvents Monodora myristica
Monodora
tenuifolia Ocimum viride
Tetrapleura
Tetrapetra
ER 3.29c±0.13 4.72a±0.014 4.25c±0.91 3.78d±0.04
AW 3.63b±0.11 3.80c±0.11 3.64e±0.71 3.91c±0.20
AN 3.67b±0.09 3.70d±0.0 4.46b±0.81 4.14b±0.11
ONE 5.18a±0.06 4.27b±0.18 16.03a±1.12 4.09b±0.12
OL 3.63b±0.02 4.33b±0.09 3.93d±0.011 4.57a±0.07
Data are means ± standard deviations (n = 3); values marked by the same letters within the same column are not significantly different (p < 0.05). ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
133
4.9.2 DPPH radical scavenging activity of Ocimum viride
Figure 7 shows dose-response curves for DPPH radical scavenging activity of five different
solvent extracts of dried ground leaves of Ocimum viride, using the DPPH colorimetric method.
Increasing concentrations (mg dry matter/ml) of extracts increased scavenging activity. The
hexane/methanol/acetone and methanol extracts exhibited the highest radical scavenging
activity (65.23 % and 65.14 % respectively), followed by acetone/water/acetic acid (62.79 %),
water (57.51 %) and acetone/hexane (57.27%) extracts. The five solvent extracts of the spice
exhibited significant difference ((p < 0.05) in radical scavenging activity. Evidently, Ocimum
viride has antioxidant compounds, and the different solvent extracts of the spice had varying
composition of antioxidant components due probably to difference in the solubility properties of
the compounds in the five solvent systems.
134
CCCCCCCCC
Concentration (mg/ml)
Fig.7. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Ocimum viride. Values are means of triplicate determinations of samples. ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v).
0
10
20
30
40
50
60
70
80
4 8 12 16 20 25Sca
ven
gin
g A
ctiv
ity
of
DP
PH
rad
ical
s (%
) ER
AW
AN
ONE
OL
135
4.9.3 DPPH radical scavenging activity of Monodera myristica
The ability of various solvents extracts of dried, ground fruits of Monodora myristica to
scavenge DPPH is compared in Fig. 8. At 10mg/ml concentration, the hexane/methanol /acetone
extract had the highest free radical scavenging ability (69.44 %), followed by water (62.0 %),
acetone/ hexane (62.05 %), methanol (60.09 %) and acetone/water/ acetic acid (62.0 %)
extracts. The DPPH radical scavenging activity of four solvent extracts of M. myristica did not
differ significantly (p > 0.05) among each other, indicating that each of the five solvent extracts
may have extracted about equal or similar antioxidant compounds. None of the five solvents
was significantly (p > 0.05) superior to the other in terms of extracting radical scavenging
antioxidant compounds.The free radical scavenging ability (19-69 %) of the various solvent
extracts were within the same range of free radical scavenging ability (7-55 %) of many
Nigerian vegetables (Oboh, 2006).
136
Concentration (mg/ml)
Fig. 8. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Monodora myristica. Values are means of triplicate determinations of samples
Concentration (mg/ml) Fig. 8. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of
Monodora myristica. Values are means of triplicate determinations. ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone/water / acetic acid (70:29.5:0.5; v/v/v).
0
10
20
30
40
50
60
70
80
90
4 8 12 16 20 25Sca
ven
gin
g A
ctiv
ity
of
DP
PH
rad
ical
s (%
) ER
AW
AN
ONE
OL
137
4.9.4 DPPH radical scavenging activity of Monodora tenuifolia
The DPPH radical scavenging activity of various solvent extracts of Monodora tenuifolia are
presented in Figure 9. The spice extracts from different extraction solvents differed significantly
(p < 0.05) in their DPPH scavenging activity which increased with increasing concentration of
the extract. The DPPH values of acetone/water/acetic acid extract were significantly higher (p <
0.05) than those of other solvent extracts of the spice within the concentration range of 8 – 25
mg dry matter / ml. Higher DPPH value indicates higher antioxidant value. The DPPH
scavenging activities were affected by the extraction solvents in the following order from high
to low: acetone / water / acetic acid > 95 % methanol > water > hexane / methanol / acetone >
acetone / hexane. This result suggests that acetone/water /acetic acid was the best extracting
solvent for DPPH antioxidant assay on Monodora tenuifolia.
138
Concentration (mg/ml) Fig. 9. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of
Monodora tenuifolia. Values are means of triplicate determinations. ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol /acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v).
0
10
20
30
40
50
60
70
80
4 8 12 20 25Sca
vengin
g A
ctiv
ity
of D
PP
H r
adic
als
(%) ER
AW
AN
ONE
OL
139
4.9.5 DPPH Radical scavenging activity of Tetrapleura tetrapetra
Figure 10 shows a dose – response DPPH radical scavenging ability of various solvent extract
of Tetrapleura tetrapetra. The free radical scavenging ability was significantly (p < 0.05) low in
acetone/hexane extract (17.03 % at 4 mg dry matter/ml and 54.49 % at 20mg dry matter/ml) and
high in water extract (28.13 % at 4 mg dry matter/ml and 79.77 % at 20mg (dry matter)/ml.
Scavenging activity of acetone/hexane extract was significantly (p < 0.05) lower than every
other extract within the concentration range in this study. The extracting solvents affected
scavenging of DPPH by the antioxidant components of the spice in the following order from
high to low: water > 95 % methanos > hexane/methanol/acetone > acetone/water/acetic acid >
acetone/hexane.
140
Concentration (mg/ml)
Fig. 10. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of
Tetrapleura tetrapetra. Values are means of triplicate determinations. ER = distilled water, OL = 95 %, methanol, ONE = acetone/hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v).
0
10
20
30
40
50
60
70
80
90
100
4 8 12 20 25Sca
veng
ing
Act
ivit
y o
f D
PP
H r
adic
als
(%) ER
AW
AN
ONE
OL
141
4.9.6 Comparison of DPPH radical scavenging activities of solvent extracts of spices
Fig. 11 compares radical scavenging activities of the four spices in the five solvents at 25mg/ml
concentrations. Tetrapleura tetrapetra exhibited highest radical scavenging activity. The
extracting solvents affected radical scavenging activities of the spices. Water extracts of T.
tetrapetra and M. myristica had highest radical scavenging constituents of both spices. Scavenging
activities (%) of the spices as affected by the extracting solvents ranged from 25 to 33.7 for O.
viride, 29.3 to 35.5 for M. myristica, 15.0 to 35 for M. tenuifolia and 38.0 to 43.0 for T. tetrapetra.
It is generally recognised that free radicals produced in the body are purely associated with
etiology of cancers and other chronic diseases. Dietary antioxidants capable of scavenging free
radicals can reduce the risks of cancer and chronic diseases (Yen et al., 1993; Duh et al., 1999).
142
SPICES SPICES
Fig. 11. DPPH radical scavenging activities of different solvent extracts (mg dry matter/mil) of Ocimum viride (O. vir), Monodora myristica (M.myr), Monodora tenuifolia (M. tri) and Tetrapleura tetrapetra (T. tetr). Values are means of triplicate determinations. ER = distilled water, OL = 95 % methanol, ONE = acetone / hexane (1:1; v/v), AN = hexane / methanol / acetone (2:1:1; v/v/v/v), AW = acetone / water / acetic acid (70:29.5:0.5; v/v/v)
0
5
10
15
20
25
30
35
40
45
O.VIR M.MYR M.TRI T.TETRSca
ven
gin
g A
ctiv
ity
of
DP
PH
rad
ical
s (%
) ER
AW
AN
ONE
OL
143
4.10 Inhibition of lipid peroxidation in cooked, ground meat patties by spices during
storage
Table 20 compares the IC50, which is the final concentration (mg/g) of the dry spice required to
suppress lipid peroxidation in cooked, ground beef and pork by 50 %, using the thiobarbituric acid
method. Higher IC50 value implies lower antioxidant activity as samples with high thiobarbituric
values had high IC50 (Table 12 and 13) (Brand-Williams et al., 1995; Sanchez-Moreno et al.,
1998. The results showed that IC50 value in pork patties ranged from 1.08 to 2.30mg/g while the
value in beef patties ranged from 1.12 to 1.79 mg / g. Ocimum viride, T. tetrapetra, M. myristica
and M. tenuifolia had lower IC50 in beef patties than in pork patties, indicating higher antioxidant
properties of these four spices in beef than in pork. At the same use level (mg / g), O. viride and T.
tetrapetra exhibited higher antioxidant properties than M. myristica and O. trifolia in both pork
and beef patties.
In lipid peroxidation, peroxides are formed and are finally decomposed to malonaldehyde as an
end product. In estimating lipid peroxidation using thiobarbituric acid (TBA) method, the
malonadehyde reacts with thiobarbituric acid (TBA) to produce red colour whose intensity
depends on the amount of malonaldehyde present. The thiobarbituric acid (TBA) method
estimated degree of lipid peroxidation in the oxidizing pork and beef by quantifying the amount of
malonaldehyde formed. The degree of deterioration of fatty foods is detected by the intensity of
red pigmentation formed when TBA is reacted with oxidizing lipid. Thiobarbituric acid method is
most sensitive and persistent than many other known methods of antioxidant analysis simply
because it quantifies the end-product which is stable for a period of time (kikuzaki et al., 2001).
144
Table 20: Inhibition of lipid peroxidation in cooked, ground meat paties by spices
IC50 ( mg / g)
Spices Pork Beef
Ocimum viride 1.80a±0.04 1.36b±0.06
Monodora myristica 2.08a±0.09 1.22bc±0.04
Monodora tenuifolia 2.30a±0.09 1.79a±0.21
Tetrapleuva tetrapetra 1.53b±0.11 1.12c±0.12
145
4.11 Comparison of Mean TBA values of meat patties treated with spices during storage
The ability of the four spices, Ocimum viride, Monodora myristica, Monodora tenuifolia and
Tetrapleura tetrapetra to inhibit lipid peroxidation in cooked, ground meat (pork and beef) during
14 days storage was estimated and the result is presented in Table 21 -24. The main effect of
treatment, storage time (1, 3, 7, 14 days), spice level (0, 0.6, 1.2, 2.0 %), the two-way interaction
between spice treatment x storage time and spice treatment x spice level were all significant at p <
0.05. The three way interaction between treatment x storage time x spice level was also significant
at p < 0.05. The spice treatment x spice level x storage time interaction of mean TBA values for
each of the spices at various concentrations in cooked ground pork and beef during 14 days
retrigerated (4 OC) storage are shown in Tables 21 - 24.
4.11.1 Mean TBA values for Ocimum viride
Table 21 shows the effect of Ocimum viride on rancidity development in cooked ground pork and
beef under storage. The mean TBA values of the cooked ground pork and beef patties that were
not treated with Ocimum viride at refrigerated storage increased to as 4.33 and 2.58
millimalonaldehyde respectively at the 14 days storage period. The samples treated with 2%
Ocimum viride had significantly (p< 0.05) lower TBA values which ranged from 1.03 for pork to
0.76 for beef at the 14th d of storage. Pork sample treated with 2.0 % Ocimum viride had mean
TBA values of 0.07 on the 4th, 1.29 on the 7th and 1.03 on the14th day of the refrigerated storage.
Conversely, pork sample with 0.6% of the spice had significantly (p < 0.05) higher mean TBA
values of 0.96 on the 4th , 2.78 on the 7th and 1.83 on the 14th while the 1.2 % treated pork sample
had 0.47 on the 4th, 1.38 on the 7th and 1.29 on the 14th days of the refrigerated storage. Pork
samples with 0.6, 1.2 or 2.0 % Ocimum viride had decreased mean TBA value of 1.83, 1.29 and
146
1.03 respectively as against the 4.33 milimalonaldehyde of the control after 14 days refrigerated
storage. Also, after 14days refrigerated storage, mean TBA value (1.03) of cooked ground pork
treated with 2.0% Ocimum viride remained significantly (p < 0.05) lower than that (4.33) of the
control sample. These results indicate that O. viride exhibited high antioxidant activity in both
cooked, ground beef and pork samples. Spices have antioxidant properties due to the presence of
phytochemical compounds such as flavonoids, terpenoids, polyphenolics, lignins and tannins
which use law activation energy to donate H+ while the resulting antioxidant radical is stable due
to delocalization of its radical election (Craig, 1999).
There was no significant difference (p > 0.05) in mean TBA values among ground beef samples
treated with 0.6, 1.2 and 2.0 % Ocimum viride. This suggests that addition of 0.6, 1.2 or 2.0 %
level of Ocimum viride to beef could achieve equal degree of lipid peroxidation inhibition in beef
and the implication is that a lower level (0.6 %) of the spice can be used toachieve the same
objective. This will save cost as well as uptimise utilization of the spice as a natural antioxidant
and flavouring ingredient.
147
Table 21: Interaction between cooked meat samples and O. viride spice levels on mean TBA values (milimalonaldehyde) during storage.
Spices/meat Spice levels Storage Time (days, 4 OC)
(% meat wt) 1 2 4 7 14
Ocimum viride
Pork 0 0.46b±0.01 0.49a±0.00 1.35b±0.04f 4.23a±0.03 4.33a±0.4
0.6 0.45b±0.01 0.48a±0.01 0.96c±0.03g 2.78b±0.12 1.83c±0.04
1.2
2.0
0.88a±0.72
0.45b±0.01
0.46a±0.01
0.46a±0.01
0.47e±0.03i
0.07f±0.00
1.38c±0.01
1.29d±0.02
1.29d±0.04
1.03e±0.11
Beef 0 0.16c±0.01 0.46a±0.01 1.86a±0.01 2.41b±0.14 2.58b±0.01
0.6 0.16c±0.00 0.17b±0.00 0.72d±0.01 0.97d±0.06 0.87e±0.04
1.2 0.15c±0.01 0.17b±0.00 0.62d±0.03 0.93d±0.00 0.81ef±0.01
2.0 0.15c±0.0 0.16b±0.00 0.63d±0.05 0.96d±0.11 0.76f±0.05
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05). wt = weight.
148
4.11.2 Mean TBA values for Monodora myristica
Table 22 compares thiobarbituric acid values of beef and pork patties treated with different levels
of African nutmeg (Monodora myristica), a populr Nigerian spice. Monodora myristica decreased
lipid peroxidation in both cooked ground port and beef patties. There were progressive decreases
in maen TBA values of both cooked ground pork and beef samples with increase in spice
concentrations and storage time when compared with the control (no spice) sample. On the 14th
day of the refrigerated storage, the control samples showed mean TBA values of 2.58 and 4.33 for
ground beef and pork respectively. Thus, the pork developed rancidity faster than the beef and
Monodora myristica suppressed rancidity faster in the beef than in the pork sample. Higher
amount of the spice would be required in pork than in beef to achieve the same level of protection
against rancidity.
149
Table 22: Interaction between cooked meat samples and M. myristica spice levels on mean TBA values (milimalonaldehyde) during storage.
Spices/meat Spice levels Storage Time (days, 4 OC)
(% meat wt) 1 2 4 7 14
Monodora
myristica
Pork 0 0.46a±0.01 0.50a±0.00 2.16a±0.04 4.27a±0.08 4.77a±0.44
0.6 0.45a±0.01 0.48a±0.01 2.03a±0.03 3.63b±0.12 4.57a±0.04
1.2 0.45a±0.04 0.48a±0.01 1.79bc±0.03 2.40c±0.01 1.99c±0.09
2.0 0.17b±0.00 0.46a±0.01 1.22c±0.00 2.06d±0.00 1.99c±0.1
Beef 0 0.18b±0.01 0.47a±0.01 1.07d±0.01 2.39c±0.13 2.58b±0.01
0.6 0.16b±0.00 0.46a±0.00 1.07d±0.01 0.89e±0.06 1.30d±0.05
1.2 0.15b±0.01 0.17b±0.00 0.32e±0.03 0.89e±0.00 1.30d±0.01
2.0 0.15b±0.01 0.16b±0.00 0.30e±0.06 0.68e±0.12 0.80e±0.05
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05). wt = weight.
150
4.11.3 Mean TBA values for Monodora tenuifolia
Monodora tenuifolia also exhibited antioxidant effect against rancidity development in cooked,
ground pork and beef. This is shown in Table 23 below. For the pork samples, mean TBA values
ranged from 3.15 to 3.52 for the spice-treated samples, and was 4.33 in the control (no spice) on
the 14th day of storage. Application of 0.6 % Monodora tenuifolia to cooked ground pork reduced
meanTBAvalue from 4.33 to 3.52 while 2 % addition reduced the mean TBA value from the same
4.33 to 3.33 in the cooked, ground beef samples. Mean TBA values of the pork samples, though
increased with storage time but decreased significantly (p < 0.05) with increase in concentration of
Monodora tenuifolia application.
Similar but higher antioxidant activity of Monodora tenuifolia was observed in treated beef
samples. Previous work on antioxidant mechanisms of spices in model systems has been identified
with various phenolic compounds that are type I antioxidants, capable of interuption of the
initation and propagation steps of lipid oxidation by donation of hydrogen (H.). In Chinese
marinated pork shanks, antioxidant effects were observed compared to controls and the effects
were attributed to star anise as a marinade ingredient (Wang et al., 1997).
However, it cannot be ruled out that the fibre component of spices may bind ionic iron in cooked
meat systems, and thus behave as type 2 antioxidants (Vasavada et al., 2006). Mean TBA values
of the spice-treated beef samples ranged from 0.91 to 1.86 as against the values (1.86 to 2.58) in
the control beef sample within the 3rd to 14th days of storage. On the 14th day of storage, the
control beef sample had significantly (p < 0.05) higher mean TBA value (4.33) than the 2%
151
Monodora tenuifolia-treated beef sample which had mean TBA value of 1.36 . The beef sample
treated with 0.6% Monodora tenuifolia had mean TBA value of 1.86 on the 14th day of
refrigerated storage. Thus, it was evident from the result that mean TBA value increased with
increase in storage time all the beef samples but significant decreases were observed with increase
in spice concentrations. The observed decreases in mean TBA values in the pork and beef samples
suggests reduced rancidity development which implies longer shelf life of the meat samples. The
spice Monodora tenuifolia exhibited appreciable antioxidant activity. It was more effective in
beef than in pork patties.
152
Table 23: Interaction between cooked meat samples and M. tenuifolia spice levels on mean TBA values (milimalonaldehyde) during storage.
Spices/meat Spice levels Storage Time (days, 4 OC)
(% meat wt) 1 2 4 7 14
Monodora
tenuifolia
Pork 0 0.44a±0.05 0.49b±0.00 2.83a±0.18 4.23a±0.09 4.33a±0.44
0.6 0.45a±0.00 0.49b±0.00 2.55b±0.01 3.10b±0.00 3.52b±0.04
1.2 0.15b±0.00 0.47b±0.00 2.46b±0.03 2.57c±0.02 3.31b±0.17
2.0 0.15b±0.00 0.44b±0.01 2.07c±0.04 2.44c±0.03 3.15c±0.06
Beef 0 0.14b±0.00 0.20cd±0.0 1.86d±0.01 2.48c±0.01 2.58d±0.01
0.6 0.15b±0.00 0.17d±0.00 1.07e±0.01 2.39c±0.01 1.86e±0.01
1.2 0.46a±0.01 0.17d±0.00 0.96e±0.02 1.48d±0.03 1.39f±0.01
2.0 0.45a±0.00 1.65a±0.01 0.91e±0.00 1.37d±0.01 1.36f±0.03
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05). wt = weight.
153
4.11.4 Mean TBA values for Tetrapleura tetropetra
Tetrapleura tetrapetra inhibited formation of thiobarbituric acid reactive substances (TBARs) in
dose-dependent manner in both cooked ground pork and beef (Table 24). When meat is cooked,
iron liberated from myoglobin interacts with phospholipids to catalyse lipid peroxidation, thereby
increasing the development of rancid flavour. The Fe-phospholipid complexes increase formation
of TBARS by catalysing the rapid autoxidation of its unsaturated fatty acid components (Lee and
Handricks, 1995). The control (no spice) pork sample had mean TBA values ranging from 0.51 to
4.28 while the pork samples treated with Tetrapleura tetrapetra had mean TBA values ranging
from 0.45 to 2.04 within the 1st to 14th days of storage. Pork samples treated with 0.6 % and 2%
Tetrapleura tetrapetra had mean TBA value of 2.04 1.28 respctively on the 14th day of storage
while the control had mean TBA value of 4.58 on the same 14th day of the refrigerated storage.
Treatment with Tetrapleura tetrapetra significantly (p < 0.05) decreased mean TBA values
throughout the storage period in the pork samples. Among the cooked ground beef samples, the
control had mean TBA values of 1.33 to 1.28 from 7th to 14th days of refrigerated storage. The
beef sample treated with 2 % T. tetrapetra had mean TBA value range of 1.81 to 0.9 from the 7th
to 14th days of storage. Most previous studies on antioxidant effects of spices have been conducted
in model systems with a few in food systems. Clove at 0.05 % was shown to enhance storage
stability and acceptability of frozen fish mince for about 28 weeks. For 50 weeks storage, an
addition of 0.1% was uptimal (Joseph et al., 1992). Clove powder at 0.2 % w/w basis significantly
(p < 0.05) reduced oxidative rancidity and improved acceptability of oysters (Abraham et al.,
1994).
154
Lowest effective spice level for Ocimum viride, Monodora myristica, Monodora trifolia and
Terapleura tetrapetra, as observed in this study, was 0.6 %. Lowest spice level is defined as the
lowest spice (mg / 100 g meat) that significantly (P<0.05) suppressed mean TBA values of the
spice-treated meat patties as compared with the control meat patties (not treated with spices)
(Jayasingh and Conforth, 2003). According to Tarladgis et al.(1960), mean TBA values > 1.0 is
usually associated with rancid flavour and odour by sensory panelists. Jayasingh and Conforth
(2003) noted that the meanTBA values of raw and cooked ground pork samples treated with milk
mineral, butylated hydroxytoluene and sodium tripolyphosphate remained less than 1.0 for the
entire 15 days storage. Similar results were evident from this study with the cooked, ground beef,
though not with the pork samples treated with 0.6 – 2.0 % O. viride, M. myristica and T. tetrapetra
(Table 21 -23).
Ground black pepper oleoresin extracted by supercritical carbon dioxide was more effective in
reducing lipid oxidation of cooked ground pork than oleoresin extracted by conventional method
(Tiprisukond et al., 1998). This is in line with the results of this stiudy supporting that the type of
solvent and in addition the methods of extraction used on the spices greatly determine the
composition and antioxidant potency of the extracts. Cinnamon essential oil has been shown to
have great antioxidant activity in Chineese style sausages (Ying et al., 1998). Ginger at 0.5% in
cooked, ground beef maintained mean TBA values less than 1.0 for 15 days refrigerated storage
(Vasavada et al., 2006; Vasavada and Conforth, 2006). Ginger extract (3 %) had been effectively
used for improving the sensory quality and shelf life of cooked mutton chunks (Mendiratta et al.,
2000). Fresh pork treated with 5 % ginger extract in combination with lactic acid (1 %), liquorice
(1%), acetic acid (1%), and garlic extract (4 %) was shown to maintain freshness for 144 h and
155
was comparable with fresh pork within 24 to 48 h of slaughter (Zhang et al., 1996). Antioxidant
effects of cinnamon, clove, fennel, pepper, star anise, ginger and black pepper in model and food
systems have been investigated (Ying et al., 1998, Dwivedi et al., 2006).
156
Table 24: Interaction between cooked meat samples and T. tetrapetra spice levels on mean TBA values (milimalonaldehyde) during storage.
Spices/meat Spice levels Storage Time (days, 4 OC)
(% meat wt) 1 2 4 7 14
Tetrapleura
tetrapetra
Pork 0 0.45a±0.00 0.51b±0.03 2.71a±0.03 4.28a±0.01 4.58a±0.01
0.6 0.45a±0.00 0.48b±0.01 2.11b±0.08 2.64b±0.11 2.04b±0.06
1.2 0.15b±0.00 1.60a±0.09 1.62c±0.02 2.01c±0.02 1.16cd±0.06
2.0 0.14b±0.00 0.45b±0.00 1.27d±0.06 1.64de±0.16 1.28cd±0.10
Beef 0 0.16b±0.00 0.17c±0.00 0.51f±0.04 1.33e±0.03 0.98d±0.02
0.6 0.15b±0.01 0.17c±0.00 0.51f±0.04 1.33e±0.03 0.98d±0.02
1.2 0.14b±0.00 0.17c±0.60 1.07e±0.12 1.29e±0.07 0.90d±0.01
2.0 0.14b±0.00 0.17c±0.00 0.65f±0.02 1.81d±0.04 0.89d±0.03
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05). wt = weight.
157
4.12 Antimicrobial activities of the spices on some selected food pathogens
4.12.1 Sensitivity of three bacteria toward inhibitory activity of aqueous and ethanol
extracts of the spices
The minimum inhibitory concentration (MIC) of aqueous and ethanol extracts of Ocimum viride,
Monodora myristica, Monodora tenuifolia and Tetrapleura tetrapetra on three test organisms in
nutrient broth is shown in Table 25. The three bacteria (Escherichia coli, Staphylococcus aureus
and Salmonella typhii) were sensitive to antirnicrobial activity of the eight spice extracts but
reacted differently to each of these extracts. The ethanol extracts showed more inhibitory activity
against the test organisms and had lower minimum inhibitory concentration (MIC) than the
aqueous extracts of most spices. However, the aqueous extracts of O. viride and T. tetrapetra had
equal MIC with the ethanol extracts of the four spices against S. aureus and S. typhii. The MICs
(mg / ml) of aqueous extracts of most of the spices were 40mg/ml against the test organisms
except that of O. viride against E. coli which was 30 mg / ml.
The MIC of the ethanol extracts ranged from 10mg/ml for O. viride against S. typhii to 40 mg / ml
of O. Viride against S. aureus and 40mg/ml of T. tetrapetra against S. typhii, showing that there
were variations in degree of antibacterial activities of the extracts against the test bacteria. The
variation could be due to differences in the active compounds present in the spices and the degree
of solubility of these active compounds in the solvents (water and ethanol). Nwinyi et al. (2009)
also reported sensitivity of both E. coli and S. aureus to antibacterial activity of aqueous and
ethanol extracts of O. viride.
158
However, with ethanol extract exhibiting MIC of 2.5 mg / ml against S aureus and 10 mg / ml
against E. coli; and aqueous extract exhibiting MIC of 10mg/ml against both E. coli and S. aureus
on nutrient agar, nutrient broth, with very high water content, may have aided viability of the
bacteria and accounted for the high MICs in the present study. Generally, this result showed that
aqueous extracts of the four spices had lower MICs than the ethanol extracts against the three test
organisms, and that the degree of antimicrobial activities of these spices to these organisms from
high to low were in the following order: Escherichia coli> Salmonella typhii > Staphylococcus
aureus. This result confirms the efficacy of these spices in traditional cuisines and cultural
medicine over the years.
159
Table 25: Minimum inhibitory concentration (MIC) of aqueous and ethanol extracts of Ocimum viride, Monodera myristica, Monodora tenuifolia and Tetrapleura tetrapetra against the test organisms.
Test spices Escherichia coli Staphylococcus
aureus
Salmonella typhii
Water
extract
(mg/ml)
Ethanol
extract
(mg/ml)
Water
extract
(mg/ml)
Ethanol
extract
(mg/ml)
Water
extract
(mg/ml)
Ethanol
extract
(mg/ml)
Ocimum viride 30 30 40 40 40 10
Monodora myri
stica
40 20 40 30 40 30
Monodora
trifolia
40 30 40 30 ND 30
Tetrapleura
tetrapetra
40 20 40 30 40 40
ND = Not determined.
160
4.12.2 Growth of pathogens in control substrate
Growth of the pathogens in subsrates was analysed using nutrient agar counts of the pathogens
Escherichia coli, Salmonella typlii and Staphylococcus aureus incubated at 37 OC for 24 h. Data
derived from nutrient broth, a general media, were used to detect potential cell injury due to
exposure to spice extracts or the food extracts. Growth of E. coli, S. typhii and S. aureus were
obtained in all control (no spice extracts) substrates (Tables 26 to 37). Growth of the three
pathogens was significantly (p < 0.05) faster in nutrient broth and beef extract than in the
vegetable and rice extracts. Within four days of storage, pathogen population increased (p < 0.05)
from 30.2 to 38.35 x 106 CFU / ml in the broth medium while in the rice and vegetable extracts,
population increases ranged from 8.8 to 18.9 x 106 CFU / ml (E. coli), 9.4 to 16.4 x 106 CFU / ml
(S. typhii), and 9.1 to 18.0 x 106 CFU / ml (S. aureus).
Within the four days of storage, pathogen populations in the beef extract were comparable (p >
0.05) to those in the nutrient broth. This might most likely be due to positive sensitivity of the
pathogens to high nutrient composition of the beef, which may be comparable to composition of
the nutrient broth. On the last (10th) day of storage, pathogen counts ranged from 35.72 to 47 95 x
106 CFU / ml in the nutrient broth (Table 35 and 36) and from 4.5 to 25.4 x 106 CFU / ml in the
vegetable and rice extracts (Table 25 and 35). This result comfirms that the extent of growth
enhancement or suppression of pathogens in food extracts vary with the nutrient composiotion
among other variables.
161
4.12.3 Antibacterial activities of Monodora myristica against food pathogens
The antimicrobial activity of M. myristica extract against E. coli inoculated in nutrient broth or
food extracts is shown in Tables 26. Monodora myristica inhibited growth of E. coli in both the
food extracts and nutrient broth. Types of substrates affected the ability of M. myristica to inhibit
growth of the pathogen. Monodora myristica at 10 mg / ml and 30 mg / ml concentions inhibited
(p < 0.05) growth of E. coli as compared to its growth in control media (broth or food extracts).
The spice inhibited growth of E. coli more in food extracts than in the nutrient broth; and the
inhibitory effect in the food extracts was in the following order from high to low: rice extract >
vegetable extract > beef extract. No detectable survivors of E. coli were obtained in inoculated rice
extract containing 30 mg / ml of M. myristica from day 4 to day 10 of storage.
162
Table 26: Mean microbial (Escherichia coli) population (106 x Cfu / ml) in aqueous food extracts (10 %) treated with different levels of Monodora myristica Substrates Spice
concentrations (mg / ml)
Storage Time ( days, 4 OC)
2 4 7 10
Nutrient 0.0 37.11b ± 3.5 38.33a± 6.36 37.92a± 13.4 40.23a ± 5.7
Broth 10 31.12c ± 10.6 29.22c ±7.78 20.56d ±7.1 14.41f±3.53
30 40.01a±2.283 18.92e± 4.22 12.0i±2.81 10.04g±4.24
Beef 0.0 22.62d ± 5.7 29.31c± 6.31 30.61b±8.48 39.44b±4.24
10 22.55d ± 7.78 30.95b±2.12 25.95c±2.12 20.92e±1.41
30 20.451e± 3.53 28.35d ± 6.36 18.71f±0.00 14.93f±2.83
Vegetable 0.0 7.90f ± 2.83 13.6f ±7.07 17.97g±4.24 23.15d±3.53
10 3.51h ± 4.20 11.95g ± 2.12 14.25h±4.94 12.52f±5.65
30 3.95h± 3.53 5.15e ± 6.34 8.6j±4.24 9.95h±2.12
Rice 0 6.41g ± 1.4 8.8h ± 1.41 23.91e±2.8 24.3c±8.51
10 7.45g ± 10.6 2.5.52f ± 4.9 3.0k±11.3 4.51i±3.52
30 1.74g± 9.81 0.00i ± 0.00 0.00l ± 00l 0.00j ± 00
Data are means ± standard deviations (n = 3); values marked by the same letter within the same column are not significantly different (p < 0.05).
163
Tables 27 shows antimicrobial activity of M. myristica extract against S. typhii inoculated in
nutrient broth or food extracts. Monodora myristica extract also exhibited higher antimicrobial
effect against S. typhii in food extracts than in broth medium. The spice did not completely inhibit
but significantly (p < 0.05) reduced growth of S. typhii in the beef, rice and vegetable extracts
containing 10mg/ml and 30mg/ml of M. myristica during 10 days storage. Survival of Salnonella
typhii was affected by the food types. Differences in growth of S. typhii in the food extracts were
more readily visualized on the 7th and 10th day of storage. The inhibitory effect of the spice extract
against S. typhii in the food extracts was in dose-dependent order and in the following order from
low to high: beef extract > rice extract > vegetable extract.
164
Table 27: Mean microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Monodora myristica Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10
Nutrient 0.0 28.0b ± 8.5 30.3b ± 5.6 38.0b ± 0.0 38.25b ± 4.9
Broth 10 28.05b ± 7.1 29.95b ± 2.1 29.95c± 2.12 31.05c ± 16.3
30 20.75e ± 7.8 26.1c ± 2.8 4.55g ± 7.8 6.05g ± 10.6
Beef 0.0 31.7a ±14.1 36.55a ± 4.9 42.05a ± 13.4 47.1a ± 12.7
10 21.3d ± 7.1 7.6e ± 2.8 7.35f ± 7.8 13.05e ± 7.1
30 26.0c ± 28.2 7.0e ± 11.3 4.75g ± 6.3 4.0h ± 2.8
Vegetable 0.0 6.3f ±56 9.0de ±1.41 12.5d ± 4.2 19.0d ± 2.8
10 3.6g ± 2.8 7.8e ± 2.8 9.7e ± 5.6 13.5e ± 8.4
30 3.05g ± 2.1 4.05f ± 2.1 7.4f ± 2.8 8.85f ± 0.7
Rice 0 7.4f ± 5.6 9.25d ± 2.1 11.9d ± 2.8 18.55d ± 4.9
10 3.2g ± 1.4 4.4f ± 2.8 7.1f ± 1.4 8.95f ± 2.1
30 3.25g ± 3.5 3.85f ± 0.7 4.15g ± 0.7 5.65g ± 4.9
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same column are not significantly different (p < 0.05).
165
The antimicrobial activity of M. myristica extract against S. aureus inoculated in nutrient broth or
food extracts is shown in Tables 28. Growth of S. aureus was obtained in all the substrates.
Growth of the pathogen was faster and reached higher levels by the end of the 10 days storage in
the spice-treated broth and food extracts than in the controls (broth and food extracts). For
example, on the 10th day of storage and at 30 mg / ml spice cocentration, S aureus population was
6.1x106 CFU in nutrient broth, 3.9x106 CFU in beef extract, 0.05x106 CFU in vegetable extract and
6.05x106 CUF in rice extract but ranged from 18.7x106 to 42.65x106 CFU in the controls. Survival
of S. aureus in the M. myristica-treated broth and food extracts was dose-dependent and decreased
with increase in the spice concentration. The 10mg/ml and 30mg/ml M. myristica concentrations
inhibited (p<0.05) growth of pathogen as compared to the controls (broth or food extracts + no
pice extract).
In most cases, bacterial (E. coli, S. typhii and S. aureus) counts in food extracts containing
Monodora myristica extract were significantly (p < 0.05) lower at the end of storage than the
initial counts. Also better bactericidal effects were obtained with the higher 30mg/ml of M.
myristica. Escherichia coli were more sensitive to the same concentration of M. myristica than S.
aureus which also was more sensitive than S. typhii. Monodora myristica suppressed the growth of
E. coli, S. typhii and S. aureus in food extract; and this calls for further investigation of its
bactericidal on these food pathogens in real food systems.
166
Table 28: Mean microbial (Staphylococcus aureus) population (106 x Cfu/ml) of aqueous food extracts (10 % w/w) teated with different levels of Monodora myristica Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10
Nutrient 0.0 28.25b ± 4.9 30.05b± 0.7 23.9b ± 12.7 38.35b ± 3.5
Broth 10 27.45c ± 7.7 29.3b ± 6.3 23.9b ± 12.7 29.35c ± 7.8
30 2.11d ± 2.8 2.67c ± 5.6 4.35g ± 4.9g 6.1h ± 11.0
Beef 0.0 34.3a ±5.6 37.1c ± 2.8 38.8a ± 12 42.65a ± 7.8
10 7.05g ± 2.1 14.15d ± 2.8 19.25c ± 4.9 12.55g ± 6.3
30 10.9e ± 1.4 3.8h ± 5.6 3.8g ± 4.2 3.9i ± 1.4
Vegetable 0.0 6.9g ±1.4 9.3e ±4.2 16.6d ± 6.3 18.7e ± 0.0
10 5.9h ± 0.0 5.9f ± 1.2 4.3g ± 1.4 2.15j ± 0.7
30 5.4h ± 4.2 3.95h ± 0.7 2.05h ± 3.5 0.05k ± 0.7
Rice 0 8.65f ± 0.7 14.05d ± 2.1 19.01c ± 2.1 20.9d ± 16.3
10 4.55i ± 2.1 8.9e ± 00 11.5.0e ± 7.1 15.75f ± 2.1
30 3.8j ± 0 4.9g ± 2.8 5.2.5f ± 3.5 6.05h ± 3.5
Data are means ± standard deviations (n = 3); values within each type of spice marked by the same letter within the same column are not significantly different (p < 0.05).
167
4.12.4 Antibacterial activities of Monodora tenuifolia against foof borne pathogens
Antimicrobial effect of Monodora tenuifolia in nutrient broth or food extracts (beef, rice and
vegetable mix) inoculated with E. coli during refrigerated storage (4 0C) is shown in Tables 29.
Monodora tenuifolia extract was effective in inhibiting growth/survival of the pathogen. The types
of substrates affected growth of E. coli during the 10 days refrigerated storage (Table 29).
Pronounced antimicrobial effect of M. tenuifolia on E. coli population was observed in food
extracts than in nutrient broth. Among the three food extracts tested, beef extract was more
supportive to E. coli growth than the vegetable extract which in turn was more supportive than the
rice extracts within the 10 days storage. Thus, Escherichia coli counts/survival was higher (p <
0.05) in vegetable extract than in rice extract. The bactericidal effect of Monodora tenuifolia in
the food extracts was observed in the following order from high to low: rice extract > vegetable
extract > beef extract. There was a highly remarkable significanit (p < 0.05) reduction in E. coli
population in the vegetable and rice extracts but the pathogen was not totally eliminated within the
used spice concentration during the 10 days storage. This suggests that at higher spice
concentrations, E. coli might be completely eliminated in the food extracts.
168
Table 29: Mean microbial (Escherichia coli) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Monodora tenuifolia Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10
Nutrient 0.0 25.6.5b ± 0.7 30.25a± 6.5 35.05a ± 2.1 39.6a ± 2.8
Broth 10 10.5.0c ± 8.2 20.9c ± 3.5 17.65d ± 4.9 14.05d ± 2.1
30 7.2.5e ± 6.3 8.95g ± 3.5 12.15h ± 0.7 11.75f ± 0.7
Beef 0.0 30.8.5a ±2.1 30.6a ± 1.4 32.25b ± 3.5 39.35a ± 7.7
10 25.7.0b ± 7. 24.5b ± 4.2 23.85c ± 3.5 20.85b ± 3.5
30 5.7.0f ± 1.4 3.1j ± 11.1 4.0k ± 2.8 4.45i ± 2.1
Vegetable 0.0 8.1.0d ±2.8 12.3f ±2.8 16.05e ± 3.5 13.9g ± 4.2
10 7.3.5e ± 6.3 18.05d ± 2.1 12.85g ± 3.5 7.65h ± 6.4
30 5.9.0f ± 4.2 5.45i ± 6.3 7.1j ± 1.5 2.7j ± 5.6
ice 0 7.1.0e ± 2.8 14.1e ± 11.3 14.8f ± 1.4 16.55c ± 7.7
10 5.3.5f ± 6.3 6.25h ± 2.1 7.85i ± 2.1 12.85e ± 0.7
30 4.8.5g ± 3.5 2.55j ± 0.7 2.6l ± 2.8 1.65S ± 3.5
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
169
Antimicrobial effects of Monodora tenuifolia in nutrient broth or food extracts inoculated with S.
typhii during 10 days refrigerated storage (4 0C) is shown in Table 30. Salmonella typhii counts in
broth and food extracts containing M. tenuifolia extracts were lower (p < 0.05) at the end than at
the beginning of the 10d storage. On the contrary, S. typhii counts continued to increase in the
control (no M. tenuifolia) nutrient broth throughout the 10 days refrigerated storage. Higher
concentrations of M. tenuifolia confered higher bactericidal effects. For example, on the 10th day
of storage of the rice extracts, the 30mg/ml spice concentration suppressed S. typhii population
from 106 x 13.5 CFU to 106 x 3.0 CFU while the 10mg/ml spice concentration suppressed the 106
x 13.5 CFU of S. typhii population to 106 x 6.25 CFU. Similar trends of antimicrobial effects of
the spice were observed in both beef and vegetable extracts during the 10 days refrigerated
storage.
Population of S. typhii continued to decrease or remained unchanged in M. tenuifolia-treated food
extracts from day 4 to day 10 indicating antibactericidal effect of the spice on the pathogen.
High lipid content in M. tenuifolia (Table 6) suggests high content of essential oils such as
monoterpenes and sesquiterpenes (Nwinyi et al., 2009) which have been shown to have
high antioxidant and antimicrobial activity (Bergonzelli et al., 2003; Dragland et al., 2003;
Iwu, 1993).
170
Table 30: Mean microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Monodora tenuifolia. Substrates Spice
concentrations (mg / ml)
Storage Time ( days, 4 OC)
2 4 7 10
Nutrient 0.0 28.25b ± 4.9 32.5a±4.2 42.9a ± 1.4 46.0a ± 11.3
Broth 10 9.85 d± 0.7 28.25b ± 4.9 26.55c ± 4.9 27.25c ± 0.7
30 8.0e ± 2.8 9.7ed ± 1.4 11.95e ± 0.7 21.05d ± 2.1
Beef 0.0 35.85a ±4.3 32.95a ± 17.6 38.05b ± 0.7 39.4b ± 2.8
10 19.25c ± 4.9 27.25b ± 4.9 16.4d ± 4.2 11.4f ± 5.6
30 18.4c ± 16.5 20.85c ± 6.3 6.65f ± 3.5 8.05g ± 3.5
Vegetable 0.0 5.9g ±4.2 13.85d ±0.7 15.5d ± 1.4 17.5d ± 4.2
10 4.9g± 1.4 8.4e ± 2.8 11.85e ± 4.9 15.25e ± 4.9
30 6.05g ± 3.5 4.7f ± 7.0 4.85f ± 0.7 4.9h ± 0.0
Rice 0 6.45f ± 2.1 11.3de ± 7.2 11.05e ± 6.8 13.5e ± 8.9
10 6.5f ± 2.8 6.1f ± 1.4 6.05f ± 2.1 6.25g ± 1.3
30 6.15g ± 3.5 5.55f ± 3.5 4.4f. ± 2.8 3.0h ± 2.8
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
171
Table 31 shows antimicrobial effects of Monodora tenuifolia in nutrient broth or food extracts
inoculated with S. aureus during 10 days refrigerated storage (4 0C). Microbial counts
(Staphylococcus aureus) increased gradually over time during the 10d refrigerated storage within
each substrate. However, with added M. tenuifolia in each substrate, S. aureus count was
comparatively low. M. tenuifolia suppressed S. aureus population in dose-dependent order in the
nutrient broth and food extracts. Antimicrobial activity of M. tenuifolia was affected by the food
substrates in the following order from high to low: vegetable > rice > beef. On the 10th day of
storage, S. aureus count was 106 x 15.75 CFU in beef, 106 x 2.0 in vegetable and 106 x 3.3 CFU
/ml in rice extrac ts treated with 30mg/ml of M. tenuifolia. At the same 30 mg / ml M. tenuifolia
concentration, it was 106 x 23.25 CFU in nutrient broth. Increasing M. tenuifolia concentration
might completely inhibit the growth of this pathogen in real food systems.
172
Table 31: Mean microbial (Staphylococcus aureus) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Monodora tenuifolia. Substrates Spice
concentrations (mg / ml)
Storage Time (days)
2 4 7 10
Nutrient 0.0 35.15a ± 7.7 37.95b±2.1 24.05b ± 3.5 47.05a ± 7.0
Broth 10 22.85c ± 2.1 20.85c ± 2.1 20.0d ± 2.5 21.7b ± 4.2
30 19.65d ± 1.4 19.6d ± 5.6 20.35d ± 2.4 23.25b ± 7.7
Beef 0.0 33.4b ±1.8 41.0a ± 1.5 47.9a ± 1.4 48.95a ± 2.5
10 6.85f ± 2.1 11.4f ± 5.6 22.35c ± 4.9 23.85b ± 3.5
30 6.05g ± 2.1 8.15h ± 2.1 15.85e ± 4.9 15.75c ± 2.1
Vegetable 0.0 8.4e ±2.1 18.0e ±2.8 19.55d ± 3.5 24.1b ± 7.1
10 6.15fg ± 0.7 4.7i ± 1.4 4.75g ± 2.1 4.35de ± 6.3
30 5.4g ± 0.0 2.9c ± 1.4 2.3.0h ± 1.4 2.0e ± 1.4
Rice 0 6.9f ± 0.0 9.7g ± 1.4 11.55f ± 2.1 17.85c ± 0.7
10 2.45h ± 4.9 3.15j ± 3.5 4.7g ± 1.4 5.9d ± 2.8
30 2.4h ± 2.8 2.95k ± 2.1 3.2h ± 0.0 3.3e ± 2.8d
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
173
4.12.5 Antibacterial activities of Ocimum viride against food borne pathogens
Table 32 shows dose-response of O. viride against E. coli inoculated in nutrient broth or food
extracts during 10 days refrigerated (4 OC) storage. The spice showed dose-dependent inhibition
against E. coli growth during the 10 days storage. Antimicrobial effect of O. virde against the
pathogen was more pronounced in the food extracts than in the broth medium; and among the
three food extracts, antimicrobial effects was more pronounced in rice and vegetable extracts than
in beef extract. Significant (P < 0.05) reduction of pathogen counts was observed during the 10
days refrigerated storage of both nutrient broth and food extracts containing 10 mg and 30 mg of
O. viride.
Ocimum viride and its extracts could serve as an alternative to synthetic chemicals in food
preservation since the phenolic and non-phenolic compounds which occur richly in this spice
(Tables 10 to 18) have been shown to exhibit inhibitory activity against pathogenic bacteria
(Cowan, 1999) .
174
Table 32: Mean bacterial (Escherichia coli) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Ocimum viride. Substrates Spice
concentrations (mg / ml)
Storage Time ( days, 4 OC)
2 4 7 10
Nutrient 0.0 34.25a ± 6.3 36.6a±7.7 39.3a ±4.2 42.5a ± 7.0
Broth 10 13.25d ± 9.1 29.4c ± 5.6 24.25c ± 4.9 26.55c ± 3.5
30 15.5d ± 2.5 15.9e ± 9.8 2.6k ± 2.8 6.05i ± 2.1
Beef 0.0 25.75b ±7.0 31.85b ± 2.1 32.55b ± 6.3 40.0b ± 1.4
10 21.3c ± 4.2 21.6d ± 4.2 14.5f ± 4.2 14.0f ± 5.6
30 21.15c ± 2.1 21.95d ± 2.1 15.9e ± 4.2 11.95g ± 0.7
Vegetable 0.0 14.4d ±7.0 16.5e ±4.2 18.1d ± 1.4 18.45d ± 9.1
10 7.35e ± 2.1 7.1g ± 3.8 9.4g ± 1.1 6.15c ± 2.1
30 6.55e ± 2.1 8.2g ± 5.6 8.2h ± 0.0 4.35j ± 7.7
Rice 0 5.35e ± 0.7 15.05f ± 2.1 16.35e ± 7.1 17.15c ± 6.3
10 5.1e ± 2.8 5.5h ± 2.8 6.15i ± 0.7 8.85h ± 0.7
30 4.85e ± 0.7 3.55i ± 3.1 3.45j± 2.2 3.6j ± 8.0
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
175
Tables 33 shows antimicrobial activity of O. viride against S. typhii inoculated in nutrient broth or
food extracts. The spice at each storage time and at a particular concentration (mg / ml) showed
varying antimicrobial activities in the different food extracts and nutrient broth. In comparison
with the controls (no O. viride), O. viride significantly suppressed S. typhii population in dose-
dependent order in the nutrient broth and food extracts.
The spice was more effective in food extracts than in nutrient broth; and among the food extracts,
it was most effective in rice extract, followed by vegetable extract and then beef extract. The
variations were presumed to be due to differences in nutrient composition of the broth and food
extracts which with the spice extracts may synergistically inhibit growth of the pathogens. In most
instances during the storage period, bacterial population continued to decrease (p < 0.05) from day
4 to 10 of storage. Thus, O. viride inhibited the growth of S. typhii, and was dose dependent and
sensitive to type of substrate and type of pathogen.
176
Table 33: Mean microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Ocimum viride. Substrates Spice
concentrations (mg / ml)
Storage Time ( days, 4 OC)
2 4 7 10
Nutrient 0.0 32.25a ± 6.3 32.55a±4.9 39.85a ± 2.1 42.6a ± 5.6
Broth 10 6.75f ± 0.7 11.9h ± 2.8 17.15e ± 4.1 19.5d ± 4.2
30 4.0g ± 11.3 14.35g ± 6.3 15.75f ± 0.7 6.05g ± 7.0
Beef 0.0 28.2b ±5.6 31.3b ± 5.6 38.05b ± 0.7 40.05b ± 0.7
10 23.5c ± 1.4 23.9d± 1.4 25.2c ± 5.6 31.9c ± 1.4
30 15.45de ±7.7 29.5c ± 2.1 5.25h ± 6.3 4.15gh ± 2.1
Vegetable 0.0 16.4d ±7.0 17.45e ±4.9 18.75d ± 2.1 20.1d ± 14.1
10 6.7g ± 0.0 8.15i ± 3.5 9.7g ± 1.4 12.8f ± 2.8
30 6.5g ± 4.2 6.0k ± 2.8 5.0hi ± 2.8 6.05g ± 27.5
Rice 0 16.25d± 6.3 16.4f ± 0.0 17.4e ± 5.6e 17.15e ± 6.3
10 9.25e ± 3.5 7.0j ± 2.8 4.55i ± 7.7 3.7h ± 1.4
30 5.0fg ± 4.2 6.1k ± 9.8 5.35h± 3.5 5.9g ± 14.0
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
177
Table 34 shows antimicrobial effects of Ocimum viride in nutrient broth or food extracts
inoculated with S. aureus during 10 days refrigerated storage (4 0C). S. aureus counts in food
extracts containing O. viride were lower (p < 0.05) at the end of the 10 days storage than at the
early stage of storage; and higher bactericidal effects were observed at higher (30 mg / 100 ml)
than at lower concentration of the spice. The spice was more effective in food extracts than in
nutrient broth probably because the broth has more balanced nutrients for S. aureu. Antimicrobial
activity of the spice against S. aureus was affected by the food extracts in the following order from
high to low: vegetable > rice > beef. No detectable survivor of S. aureus was found in the
vegetable extract containing 30mg/ml of O.viride from day 4 to day 10 of storage.
Based on the survival counts of the pathogens from day 4 to day 10, the spice O. viride seemed to
be more effective against S. aureus than against E. coli and S. typhii (Tables 32 - 34). Its
antibacterial effects against these pathogens were in the following order from high to low: S.
aureus > E. coli > S. typhii.
178
Table 34: Mean microbial (Staphylococcus aureus) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Ocimum vivide. Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10 Nutrient Broth 0.0 19.2b ± 63.6 35.15b ± 3.5 41.9a± 35 47.45a± 7.7
10 18.6bc.± 9.8 18.85c ± 0.7 18.1c± 1.4 15.35c ± 3.5
30 15.0d± 4.2 13.0e± 1.4 11.10d ± 2.8 9.2.0e± 8.4
Beef 0.0 22.85a± 13.4 36.9a± 12.7 37.6b± 2.8 4.32.0b ± 0.0
10 16.15c ± 10.6 15.05d ± 12.0 9.65e± 3.5 8.65e± 7.7
30 14.7d ± 1.4 12.6e± 8.4 8.45f ± 6.3 5.05f ± 3.5
Vegetable 0.0 9.55e ± 2.1 9.1f ± 1.4 10.05e± 2.1 14.4d ± 2.8
10 6.85fg ± 7.7 6.85g± 0.7 5.2g ± 2.8 41.0g ± 4.2
30 0.15i ± 2.1 0.0j ± 0 0.0i ± 0 0.0i ± 0
Rice 0.0 7.05f ± 2.1 9.7f ± 1.4 5.6g ± 8.4 4.5g ± 4.2
10 4.55g± 17.6 5.7h ± 1.4 11.0e± 8.4 4.5g ± 4.2
30 3.3h± 8.4 3.45i ± 3.5 2.85h± 0.7 3.05h ± 9.1
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
179
4.12.6 Antibacterial activities of Tetrapleura tetrapelra against food borne pathogens
The relative antibacterial activities of T. tetrapetra against E. coli in nutrient broth and food
extracts stored for 10 days at 4 OC is shown in Tables 35. T. tetrapetra inhibited the growth E. coli
in both nutrient broth and food extracts. The inhibitory effect of this spice against E. coli was most
effective in vegetable, and was followed by rice, beef and then nutrient broth. No survival/growth
of E. coli was detected in vegetable extract containing 30 mg / ml of T. tetrapetra from day 4 to
day 10 of storage.
Escherichia coli, a gram negative organism, has high content of lipids which is believed to
prevent injurious materials from reaching the active site of action within the cells (Kim et
al., 1995; Kim et al., 2006). This spice was abls to destroy this defensive mechanism of E.
coli and then penetrate its inner cell to completely inactivate the patogen.
180
Table 35: Mean microbial (Escherichia coli) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Tetrapleura tetrapetra. Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10 Nutrient Broth 0.0 19.4c ± 5.6 30.2b ± 7.0 32.1b ± 1.4 35.75b ± 2.1
10 11.95d ± 2.1 27.2c ± 14.1 17.25de ± 6.5 8.75f ± 2.1
30 11.25d ± 7.1 20.7d ± 8.4 7.8h ± 1.4 8.05g ± 2.1
Beef 0.0 22.65b± 4.9 33.8a ± 11.3 38.0a ± 9.8 51.45a ± 7.1
10 33.5a ± 9.8 14.25f ± 16.6 16.55e ± 4.9 14.3d ± 9.8
30 11.45d ± 6.3 20.7d ± 7.7 14.7f ± 1.4 7.0h ± 1.4
Vegetable 0.0 9.05e ± 3.5 18.9e ±2.8 20.75c ± 9.8 25.4c ± 2.8
10 2.6g ± 2.8 2.95j ± 2.1 7.6h ± 9.8 7.55h ± 3.5
30 2.35g ± 7.7 0.00k ± 0 0.00j ± 0.0 0.00j ± 0.0
Rice 0.0 6.85f ± 0.7 9.0g ± 1.4 11.0g± 2.8 12.85e ± 3.5
10 6.55f ± 4.9 6.2h ± 5.6 3.45i± 3.5 3.15i ± 0.7
30 2.75g ± 4.9 3.45i ± 2.1 3.55i ± 3.5 3.15i ± 0.7
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
181
The effect of Tetrapleura tetrapetra on growth/survival of S. typhii in nutrient broth or food
extracts stored for 10 days at 4 OC is shown in Table 36. Tetrapleura tetrapetra inhibited growth
and survival of this pathogen in both nutrient broth and food extracts.The ability of this spice to
inhibit microbial growth was affected by the type of pathogens, type of substrates and the spice
concentration in these substrates. Just like the cases of M. myristica, M. tenuifolia and O. viride, its
antimicrobial effects on S. typhii were more pronounced in food extracts than in nutrient broth.
However, out of the three food extracts, beter antibacterial effect was observed in rice and
vegetable extracts than in beef extract. The antimicrobial effects of the spice on the pathogens in
rice and vegetable extracts were influenced (p < 0.05) by both its extract concentrations and
pathogen strain. Bacterial counts in food extracts containing T. tetrapetra extract were lower (p <
0.05) at the end of storage than at the initial stage of storage. Also, better bactericidal effects were
observed at higher (30 mg / ml) spice concentration. In all the food extracts treated with T.
terapetra, S. typhii population decreased (p < 0.05) from day 4 to day 10 of storage; except in few
cases where the decrease was not continual.
182
Table 36: Mean microbial (Salmonella typhii) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Tetrapleura tetrapetra. Substrates Spice
concentrations (mg/ml)
Storage Time (days)
2 4 7 10 Nutrient Broth 0.0 25.4b ± 2.8 31.85b ± 2.1 42.6a± 5.6 47.95a± 0.7
10 19.35c ± 6.3 21.95c ± 2.1 25.8d± 6.3 38.6c ± 0.0
30 16.25d ± 9.1 21.9c ± 1.4 13.7f ± 1.4 13.35g ± 6.3
Beef 0.0 26.85a± 4.7 32.35a± 6.3 37.7b± 4.2 45.2b ± 1.0
10 14.45e± 12.0 19.1d ± 7.0 35.65c ± 7.7 38.05d ± 7.7
30 14.95e± 4.9 10.2.0g ± 5.6 8.9h ± 4.2 5.55l ± 4.9
Vegetable 0.0 11.7f ± 1.4 14.05f ± 3.5 17.0e± 2.8 25.42e± 2.8
10 7.9g ± 1.4 9.35h ± 6.3 11.1g± 1.4 18.95f ± 10.0
30 1.75j ± 4.2 2.8k ± 8.4 4.75j ± 4.9 12.2h ± 5.6
Rice 0.0 15.25d ± 0.3 15.9e± 7.0 16.85e± 6.3 6.6k ± 4.2
10 6.65h± 4.2 7.75i ± 0.7 8.95h± 0.7 9.3i ± 5.6
30 4.0.5i ± 0.7 5.0j ± 2.8 6.5i ± 2.8 7.5j ± 4.2
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
183
Table 37 shows the antimicrobial effects of T. tetrapetra in nutrient broth or food extracts
inoculated with S. aureus during 10 days storage at 4 OC. T. tetrapetra inhibited the growth of S.
aureus in both the nutrient broth and food extracts in dose-dependent order, and more inhibitory in
food extracts than in the nutrient broth. The antimicrobial effect of T. tetrapetra extract against S.
aureus was more pronounced in rice extract than in vegetable extract in which it was more
pronounced than in the beef extract. Probably, because the beef extract is high in most food
nutrients that boost the growth of the pathogens despite the spice effects. Slight marginal (p <
0.05) increases were observed in microbial counts towards the end of the 10 days storage in in all
the food extracts treated with T. tetrapetra. This suggests that the spice concentrations (10 mg / ml
and 30 mg / ml) used in this study was low to furnish sufficient antimicrobial components to
completely kill the entire S. aureus population within the 10 day storage period. Therefore, higher
doses of T. tetrapetra extract than as used in this study might be sufficient for complete control of
the pathogen in foods and food extracts. Further studies are needed to address the possible use of
this spice as antimicrobial agents in real food systems.
Variation in degrees of antimicrobial activities of spices could also be attributed to differences in
composition of phytochemical constituents in the spices. The phytochemicals alkaloids, tannins,
saponins, phenols, flavonoids and oxalates are important constituents of these spices as evident
from the results of Tables 10 to 18. Flavonoids exhibited antioxidant activity and are effective
scavengers of superoxide anoins (Robak and Gryglewsk, 1988). This could significantly affect
the cell wall of S. aureus and may invariably lead to collapse of cell wall and affect the overall
mechanism of the organs.
Antibacterial effect of T. tetrapetra against the test pathogens can be represented in the order from
high to low E. coli > S. aureus > S. typhii. Antimicrobial effects of the four spices (Ocimum viride,
184
Monodora myristica, Monodora tenuifolia or Tetrapleura tetrapetra) on the three pathogens (E.
coli, S. aureus and S. typhii) in the three food extracts (beef, rice and vegetable) were better than
that obtained in the broth medium.
185
Table 37: Mean microbial (Staphyloccocus aureus) population (106 x Cfu/ml) of aqueous food extracts (10 %) treated with different levels of Tetrapleura tetrapetra. Substrates Spice
concentrations (mg / ml)
Storage Time (days, 4 OC)
2 4 7 10 Nutrient Broth 0.0 23.9b ± 11.3 33.6b ± 8.4 36.9b± 12.7 42.55a± 6.3
10 13.75c ± 6.3 17.6c ± 8.4 23.65c ± 6.3 28.25c ± 4.9
30 11.05d ± 0.4 14.25e± 6.3 20.95d ± 0.7 22.5e± 4.2
Beef 0.0 34.5a± 2.8 39.45a± 19.0 37.95a± 0.7 40.95b ± 13.4
10 1.10d ± 12.7 13.9e± 2.1 18.1e± 1.4 18.75f ± 2.1
30 9.1e± 1.4 11.0f ± 0.7 14.75g ± 2.1 18.75f ± 2.1
Vegetable 0.0 9.45e ± 6.3 17.6c ± 5.6 18.75e± 12.0 15.3g ± 4.2
10 7.2f ± 1.4 8.5g ± 5.6 11.4h± 8.4 24.9d ± 2.0
30 6.8f ± 1.4 8.7g ± 1.4 9.5i ± 4.2 14.65h ± 7.7
Rice 0.0 11.85d ± 3.5 15.1d ± 7.0 16.55f ± 4.9 17.95g ± 2.1
10 5.85g± 3.5 6.9h ± 2.8 8.6j ± 4.2 12.2i ± 2.8
30 4.5h ± 4.2 5.35i ± 0.7 7.9j ± 4.0 9.35j ± 5.6
Data are means ± standard deviations (n = 3). Values marked by the same letter within same column are not significantly different (p < 0.05).
186
Generally, composition of food materials, pH and bacterial population in the food system affect
survival of bacteria on food systems at refrigerated temperatures (D’ Aoust et al., 1982).
Treatment of inoculated nutrient broth or food extracts (beef, vegetable mix and rice) with
Ocimum viride, Monodora myristica, Monodora tenuifolia or Tetrapleura tetrapetra may injure
bacterial structures so that storage at refrigerated temperature synergistically enhances bactericidal
effects since bacteria are generally sensitive to sudden change in temperature (Kim et al., 1995).
The four spices acted differently in their ability to inhibit survival and growth of the three
pathogens in both broth and food extracts. Okigbo et al. (2005) reported that the ability of plant
extracts to inhibit microbial growth may be affected by age of the plant, extracting solvent,
methods of extraction and time of harvesting the plant materials.
Due to differences in genetics, agronomical practices and environmental factors, composition of
critical compounds in herbs and spices differ signiticantly; and as a result may exhibit varying
differences in antibacterial efficacy against foodborne pathogens (Hao et al., 1998; Chau et al.,
1999). Also, because plant extracts exhibit higher antimicrobial activity in acidic environments
(Chau et al., 1999), lowering pH of the food/food extracts might enhance inhibitory activities of
the spices/spice extracts against the food pathogens. This suggests that such plant extracts could
work better in acid foods and in combination with food grade organic acids. This calls for
researches in such direction for broather application.
187
Antimicrobial agents with low bacteriocidal/bacteriostaic activities against bacterial strains would
have high MICs, and allow high microbial counts. Each of the extracts from Ocimum viride,
Monodora myristica, Monodora tenuifolia and Tetrapleura tetrapetra exhibited high and low
degrees of MICs against E. coli, S. typhii and S. aureus (Table 26) and this indicates different
mode of action of the extracts against the pathogens. Most antimicrobial agents exert antimicrobial
action by interfering with the phospholipid bilager of the cell membrane, causing increased
permeability and loss of cellular constituents (Abee et al., 1991; Smid and Gorris, 1999);
impairing a variety of enzyme systems, including those involved in the production of cellular
energy and synthesis of structural components (Connor and Beuchat, 1984); and/or inactivating
and destroying genetic material. Using spice ingredients for inhibition of food pathogens may find
a wider application in food processing, preservation, and promotion of health of consumers.
4.12.7. Phytochemical composition of cooked spice-treated food extracts
Table 38 shows non – phenolic photochemical composition of cooked food extracts [10 % (w/w)]
treated with 2.5 % (10 mg / ml) and 7.5% (30 mg / ml) of O.viride, M. myristica, M. tenuifolia and
T. tetrapetra. Phytate, Alkaloid, oxalate and saponin earlier recorded (Tables 10 - 13) in theses
spices were also present in the cooked spice-treated food extracts and even in the controls (without
spice). Phytate (mg / 100 g) ranged from 0.064 in control pork to 2.94 in rice treated with 7.5 % O.
viride, alkaloid (mg / 100 g) from 0.003 in control pork to 9.01 in pork treated with 7.5 % M.
myristica, oxalate (mg / 100 g) from 0.023 in control pork to 9.21 in beef treated with 7.5 % O.
viride, and saponin (mg / 100 g) from 0.04 in control beef to 2.31 in vegetable treated with 7.5 %
M. tenuifolia. These phytochemicals were lower in these food extracts than in the spices.
188
Vegetable extract had highest content of these chemicals, followed by rice, beef and pork. Thus,
food types also influenced the photochemical contents. The control (without spices) beef and pork
sample had significantly (p < 0.05) low contents of these chemicals. In most of the food extracts,
phytochemical contents increased slightly with high increase, (from 10 mg/ml to 30 mg / ml spice
concentration, 75 % increase), of spices. One would presume at this point that spices have
maximum thresholds for photochemical contents in food. This could be attributed to low contents
(2.5 % and 7.5 %) of these spices in the food and also the likely effect of heat in destroying part of
these phytchemicals (Rhodes, 1996). Phytochemicals are plant metabolites and are richly found in
fruits and vegetables. Presence of these photochemical in cooked food extracts confirms that they
contribute to preservative and health promoting quality of menu.
189
Table 38: Non-phenolic phytochemical contents of water extracts of cooked spice-treated foods Food/spices Spice levels
( mg /100 g) Alkaloid (mg /100 g)
Oxalate (mg / 100 g)
Saponin (Mg /100 g)
Phytate (Mg /100 g)
BEEF O. viride 10 0.943d ±0.02 4.093b±0.07 0.544b±0.01 0.639b±0.00 30 1.763c ±0.02 9.218a±0.12 0.772a±0.03 0.773ab±0.03 M. myristica 10 1.937c±0.08 2.274c±0.06 0.257c±0.03 0.076e±0.00 30 1.981c±0.20 3.880b±0.04 0.514b±0.0 0.460c±0.00 T. tetrapetra 10 0.813d±0.08 1.490d±0.10 0.257c±0.01 0.774ab±0.02 30 3.775a±0.03 1.495d±0.01 0.514b±0.02 0.940a±0.03 M. tenuifolia 10 1.485c±0.31 3.558b±0.02 0.257c±0.00 1.165a±0.02 30 2.252b±0.02 0.779e±0.01 0.772a±0.01 0.064d±0.01 Control 0.00 0.037e±0.00 N.D. 0.043d±0.00 0.070d±0.00 VEGETABLE M. myristica 10 2.710d±0.07 5.830b±0.13 0.544c±0.02 1.439b±0.04 30 3.252c±0.02 8.974a±0.11 0.772b±0.03 2.242a±0.06 O. viride 10 2.195e±0.06 5.976b±0.08 0.557c±0.01 2.711a±0.05 30 7.2711a±0.30 8.926a±0.12 0.772b±0.01 2.711a±0.10 T. tetrapetra 10 2.375e±0.01 5.386b±0.06 0.772b±0.01 2.038a±0.03 30 2.710d±0.05 5.716b±0.02 0.772b±0.00 2.465a±0.02 M. tenuifolia 10 2.671de±0.05 5.813b±0.04 0.772b±0.00 1.186b±0.01 30 4.065b±0.02 8.029a±0.10 2.315a±0.01 2.390a±0.02 0.00 0.489e±0.01 1.773c±0.05 0.579c±0.01 1.106b±0.04 PORK M. myristica 10 1.438d±0.01 5.376c±0.09 0.257d±0.00 1.186b±0.05 30 9.014a±0.02 7.429a±0.11 0.772b±0.02 1.076c±0.01 O. viride 10 0.884e±0.03 4.236cd±0.10 0.243d±0.03 0.768d±0.01 30 2.239c±0.10 4.856cd±0.03 0.772b±0.01 0.774d±0.03 T. tetrapetra 10 1.42d±0.02 3.557d±0.07 0.257d±0.01 1.838a±0.06 30 4.136b±0.10 5.635c±0.02 1.543a±0.01 1.816a±0.00 M. tenuifola 10 1.914dc±0.01 1.5824e±0.01 0.772b±0.02 0.170b±0.01 30 4.673b±0.05 6.155b±0.04 0.514c±0.02 1.140b±0.01 Control 0.00 0.003 f±0.00 0.023f±0.00 0.514±0.03 0.064 e±0.00 RICE M. myristica 10 1.084b±0.00 2.593b±0.00 0.510c±0.02 1.897c±0.11 30 1.562b±0.02 7.015a±0.03 0.772b±0.04 2.224b±0.10 O. viride 10 1.691b±0.10 1.558c±0.01 0.257d±0.01 1.962c±0.04 30 2.168b±0.11 5.456a±0.31 0.772b±0.07 2.937a±0.01 T. tetrapetra 10 10.295a±0.20 2.598b±0.07 0.772b±0.03 2.156b±0.05 30 12.194a±0.31 6.353a±0.21 1.713a±0.08 2.560b±0.03 M. tenuifolia 10 1.350b±0.02 1.132c±0.08 0.257d±0.02 1.988c±0.10 30 2.943b±0.02 3.377b±0.10 1.543a±0.03 2.384b±0.30 Control 0.00 0.466c±0.04 1.015c±0.00 0.529c±0.01 1.330d±0.07 Values are means of three determinations±standard deviations, N.D. = Not determined.
190
Table 39 shows phenolic phytochemical profile of cooked food extracts (10 % W/W) treated with
2.5 % (10mglml) and 7.5 % of O. viride, M. myristica, M. tenuifolia and T. tetrapetra. Total
phenol, tannin and flavonoid which were very prominent in the spices were also found in the
cooked spice-treated food extracts. These phytochemicals were lower in the cooked spice-treated
food extracts than in the spices (Tables 14- 18). This could be due to low contents (2.5 % and 7.5
%) of these spices which were the main source of these chemicals in the food and probably due to
the effect hearting on the phytochemicals during cooking (Onwuka, 2005). In the spice samples,
total phenol contents (Table 14) were very much higher than tannin contents (Table 15) but tannin
contents in these food extracts (Table 39) were as high as or even higher than phenol contents.
There may be an inter conversion between these two important highly related compounds during
cooking. Also heating did not completely destroy these novel chemicals. As high as 75 % (10-30
mg / ml) increases of spices in the food samples resulted only in marginal increases of
phytochemical contents. Total phenol content ranged from 0.18 in control beef to 2.13 in pork
treated with 7.5 % of O. viride while tannin content ranged from 0.048 in control beef to 1.03 in
vegetable treated with 7.5 % M. tenuifolia. Total phenol, tannin and flavonoid contents were
highest in cooked vegetable extract, followed by rice, beef and pork extracts. Phytochemicals are
plant metabolites; fruits and vegetables are good sources of total phenol, tannin and flavonoid
(Roesler et al., 2006).
191
Table 39: Phenolic phytochemical contents of water extracts of cooked spice-treated foods Food/spices Spice treatment
mg /100 g Total phenol (GAE/100 g)
Total tannin (TAE /100 g)
Total flavonoid (GAE/100 g)
BEEF O. viride 10 0.936c±0.01 0.802a±0.01 0.022a±0.01 30 1.461b±0.02 0.849a±0.00 0.024a±0.00 M. myristica 10 0.536d±0.01 0.639c±0.06 0.004cd±0.00 30 0.605c±0.00 0.712bc±0.01 0.004cd±0.01 T. tetrpetra 10 0.913c±0.06 0.895a±0.02 0.006c±0.00 30 2.123a±0.10 0.877a±0.03 0.016b±0.00 M. tenuifolia 10 0.491e±0.01 0.583cd±0.02 0.003d±0.00 30 0.628d±0.01 0.683c±0.02 0.019b±0.00 Control 0.00 0.183f±0.05 0.048e±0.00 0.001e0.00 VEGETABLE M. myristica 10 0.742c±0.01 0.801c±0.10 0.005e±0.00 30 0.753c±0.02 0.855b±0.01 0.011c±0.00 O. viride 10 0.936b±0.02 0.936a±0.01 0.024bc0.00 30 1.335a±0.01 1.022a±0.00 0.038b±0.01 T. tetrapetra 10 0.913b±0.03 0.873b±0.06 0.017c±0.00 30 1.061ab±0.00 0.913a±0.05 0.10a±0.00 M. tenuifolia 10 0.639d±0.02 0.885bc±0.06 0.009d±0.00 30 0.684d±0.03 1.034a±0.02 0.011c±0.00 Control 0.00 0.628d±0.01 0.795c±0.02 0.002f±0.00 PORK M. myristica 10 0.502d±0.02 0.772c±0.01 0.005c±0.01 30 0.696d±0.02 0.771c±0.01 0.005c±0.01 O. viride 10 0.993c±0.03 0.927a±0.02 0.013a±0.01 30 2.134a±0.07 0.998a±0.02 0.017a±0.00 T. tenuifolia 10 1.039bc±0.04 0.986a±0.07 0.008b±0.01 30 1.495b±0.03 0.999a±0.03 0.014a±0.00 M. tenuifolia 10 0.559d±0.01 0.837b±0.02 0.003d±0.00 30 0.696d±0.00 0.909a±0.03 0.004d±0.00 Control 0.00 0.183e±0.01 0.049c±0.01 0.003d±0.00 RICE M.myristica 10 0.377c±0.01 0.640c±0.01 0.006c±0.00 30 0.764b±0.02 0.927ab±0.02 0.006c±0.00 O. viride 10 0.981a±0.04 0.885b±0.02 0.022a±0.00 30 0.776b±0.03 0.993a±0.03 0.027a±0.00 T. tetrapetra 10 N.D. 0.885b±0.03 0.009c±0.00 30 1.118a±0.05 0.933ab±0.02 0.017b±0.00 M. tenuifolia 10 0.513b±0.05 0.903a±0.01 0.013bc±0.00 30 0.616b±0.05 0.956a±0.07 0.004d±0.00 Control 0.00 0.218d ±0.01 0.933ab±0.11 0.004d±0.00 Values are means of three determinations±standard deviations, N.D. = Not determined.
192
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1. Conclusion
� The four Nigerian spices possess varying levels of different phytochemical constituents.
� Spices and solvent extracts of spices differed significantly (p < 0.05) in phytochemical
contents. The amounts of phytochemicals extracted were influenced by the solvent
systems used.
� Methanol was the best while acetone / hexane (1:1, v/v) the worst extracting solvents for
these phytochemicals.
� However, none of the solvents was consistently good in extracting a particular
phytochemicals from the spices; suggesting that spice morphology and composition might
have influenced extracting capacity of the solvents.
� Amount of phytate and flavonoid extracted were not significantly (p >0.05) affected by
solvent types used.
� Phytate was the most while anthocyanin was the least abundant phytochemicals in the
spices
� M tenuifolia had the highest alkaloid (6.54 mg/100 g), oxalate (7.0 mn/100 g) and phytate
(5.5 mg/100 g) contents, and M. myristica the highest saponin (0.719 mg/100 g) content.
� . T tetrapetra had the highest total phenol (15.93 g GAE/g), M. myristica the highest
anthocyanin (0.174 g GAE/g) and O. viride the highest condensed tannin (0.20 g TAE/g),
flavonoid (0.28 g GAE/g) and carotenoid (0.93 g GAE/g) contents. Spices were evidently
rich in most of the phytochemicals screened.
193
� Solvent extracts of spices exhibited dose-dependent antioxidant capacities in reducing Fe3+
to Fe2+, scavenging DPPH radical and in inhibiting linoleic acid peroxidation in oxidizing
FTC systems.
� As radical scavengers, these spices could be used to manage radical-related degenerative
diseases in vivo.
� Water extracts of O. viride and T. tetrapetra, methanol extract of M. tenuifolia and
acetone/water/acetic acid extract of M. myristica exhibited highest inhibition of linoleic
acid peroxidation in FTC oxidizing systems.
� Spices inhibited rancidity development in cooked ground beef and pork patties during
storage, indicating antioxidant capacities in model food systems.
� Antioxidant capacity of the spices was in the following order from high to low: T.
tetrapetra > O. viride > M. myristica > M. tenuifolia.
� Spices also exhibited dose-dependent antibactericidal effects against E. coli, S. typhii and
S. aureus; and was more pronounced in food extraxts than in broth medium, and in rice and
vegetable extracts than in meat extracts
� Exposure of pathogens to both tested levels (10 and 30 mg/ml) of spices in broth medium
and food extracts generally suppressed (p < 0.05) population compared to the control (no
spice).
� Antioxidant and antimicrobial capacities of the spices could be due to the phytochemicals.
� The spices can be exploited as natural antioxidants and antimicrobials in real food and
food-related systems and possibly for ameloriating oxidative-related stresses in vivo; and
as ingredients of functional foodS.
194
5.2. Recommendations
� Individual phytochemicals responsible for antioxidant and antimicrobial activities in these
spices should be characterised and identified;
� Modes of antioxidant and antibacterial activities of these phytochemicals should be analysed
and established for broader application in real food systems;
� The best extracting solvents for the individual phytochemicals in the spices should be
established for individual and synergistic uses in food and food-related systems;
� The spices should be exploited as natural antioxidants and antimicrobials in real food
systems.
195
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APPENDICES
Appendix 1: Preparation of reagents for phytochemical determinations
1. Aluminium (iii) Chloride solution . Twenty gram (20 g) of aluminium (iii) Chloride
hexahydrate (AlCl3.6H2O) is dissolved and admixed in 1000ml methanol.
2. Standard 2,6-dichloroindophenol. A 0.5 g of 2,6-dichloroindophenol was dissolved in 1000
ml of ditilled water.
3. 20 % glacial acetic acid. Glacial acetic acid (200 ml) is mixed with 800 ml of distilled water to
give 1000 ml of 20 % glacial acetyic acid.
4. 200mM Sodium phosphate buffer (pH=6.6). Exactly 32.8 g of anhydrous sodium phosphate
(Na2PO4) is dissolved in 1000 ml of deionised water.
5. 75 % ethanol. Absolute ethanol (750 ml) is gently added to 250 ml of distilled water in a 1Litre
holding flask.
6. 0.3 % ammonium thiocyanate solution. Ammonium thiocyanate crystals (0.5 g) are dissolved
in 1000 ml deionised water and mixed thoroughly.
7. 2 % AlCl 3.6H2O sotution. Ten grams of AlCl3.6H2O is dissolved in 500 ml of methanol and
swirled gently to mix thoroloughly.
8. 0.1 M FeCl3 in 0.1N HCl. Hydrochloric acid (2 ml) is added dropwise to 198 ml of deionised
water, mixed gently and allowed to cool before adding 0.335 g of FeCl3 crystals. This was
mixed gently and then vigorously to mix thoroughly.
9. Saturated sodium carbonate solution. A 750 g of anhydrous sodium carbonate (Na2CO3) is
dissolved in 1000 ml of distilled water and shaken vigorously to mix thoroughly.
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Appendix 2: Preparation of reagents for antioxidant determinations
1. Linoleic acid solution. A 25 ml of linoleic acid is admixed with 995 ml of absolute ethanol and
5 ml of deionised water.
2. 20 mM ferrous chloride in 3.5 % (v/v) hydrochloric acid (HCl). First, 35 ml of concentrated
HCl is added to 965 ml of distilled water, and 25.6 g of the ferrous chloride (FeCl2)
dissolved in the mixture with continuous stirring to mix thoroughly.
3. Standard solution of ascorbic acid. Ascorbic acid (25 g) is dissolved in 500ml of distilled
water, mixed thoroughly and filtered through No5 Whatman filter paper.
4. Standard iron (iii) chloride (35 mM FeCl3) solution. Exactly 5.758 g of FeCl3 is dissolved in
1000ml of deionised water, shaken vigorously to mix thoroughly.
5. 30 % Ammonium thiocyanate. Ammonium thiocyanate crystal (150 g) is dissolved in 500 ml
of deionised water.
6. Thiobarbituric acid reagent. Fifteen milliliter each of concentrated hydrochloric acid (HCl)
and trichloroacetic acid (TCA) were admixed with 70ml of distilled water; and 0.375 g of
thiobarbituric acid dissolved in the resulting solution.
7. 1, 1 Diphenyl -2- picryl hydrazyl (DPPH) reagent. Exactly 4 mg of DppH was dissolved in
100 ml of absolute ethanol in the dark.
8. Potassium ferricyanide reagent. Exactly 2 g of potassium ferricyanide was dissolved in
200 ml of deionised water.
9. Trichloroacetic acid reagent. Two grams of Trichloroacetic acid was deionised in 200 ml
of distilled water.
10. Iron (III) Chloride (Fecl 3) reagent. Exactly 0.2 g of FeCl3 was dissolved in 200 ml of
deionised water.
214
Appendix 3: Preparation of reagents for antimicrobial screaning
1. Preparation of nutrient agar. Weighed 28 g of nutrient agar powder was carefully dispersed
in 1 litre of deoinised water, allowed to soak for 10 min. and then swirled to mix
thoroughly. This was sterilized by autoclaving at 121 OC for 15 min., cooled to 45 OC,
mixed well and then dispersed aseptically into petri dishes. These were allowed to
set before use for antimicrobial activity.
2. Preparation of nutrient broth . Weighed 13 g of nutrient broth powder was carefully dispersed
in 1000 ml of distilled water, allowed to soak for 10 min. and then swirled to mix
thoroughly. This was sterilized by autoclaving at 121OC for 15 min., cooled to 45 OC,
mixed thoroughly and then dispersed aseptically into sterile bottles. These were allowed to
cool before use for antimicrobial screening.
3. Preparation of Macfarland 0.5 turbidity standard (NCCLS, 2006). The stock solution of
Macfarland 0.5 turbidity standard is prepared from two stock solutions, A and B.
Solution A (0.048 M BaCl2) is prepared by admixing 1.75 g of BaCl2.2H2O to 50 ml of distilled
water and then making up to 100 ml with distilled water in 100 ml bottle.
Solution B (0.36N H2S04) is prepared by adding 1.0 ml of concentrated sulphuric acid (Analar
grade, Specific gravity of 1.84) into 50 ml of distilled water and making up to 100 ml with
distilled water in 100 ml bottle.
Stock solution is prepared by adding 0.5ml of solution A into 99.5 ml of solution B in 100 ml
transparent bottle. Note that a bacterial suspension in distilled water that has the same
turbidity as the stock solution is assumed to contain 105 CFU of the bacteria.
