PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF FOUR NIGERIAN SPICES
ABSTRACT
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.
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
Phytoestrogens18
Phytochemical metabolism in human18
Lipid oxidation19
Degenerative effects and suppression of lipid oxidation ……………….21
Functions and mechanism of action of antioxidants in foods …………23
2.15 Natural antioxidants …………………………………………………….. 24
Antioxidant properties of spices and spice extracts ……………………...25
Assessment of antioxidant activity of antioxidant compounds and the degree
of lipid Oxidation in food system………………………………………………... 26
Major microorganisms of food poisoning ……………………………….28
Important preservation techniques for preventing food poisoning from
pathogenic microorganisms ………………………………………………. 28
Antimicrobial properties of spices and spice extracts ……………………30
Choice of solvents for preparation of crude extracts from biological Materials31
Biology and ecology of Tetrapleura tetrapetra (Schum & Thonn)34
Nutrient composition of Tetrapleura tetrapetra34
Food and medicinal uses of Tetrapleura tetrapetra35
Ecology, botany and distribution of Monodora tennifolia (Benth)36
Chemical composition and uses of Monodora tennifolia (Benth)36
Ecology, botany and distribution of African nutmeg (Monodora myristica Gaetn)37
Chemical composition and uses of African nutmeg
(Monodora myristica Gaertn) …………………………………………….. . 37
Ecology, botany and distribution of Ocimum viride (Willd) ……………...38
Chemical composition and uses of Ocimum viride38
CHAPTER THREE
3.0 MATERIALS AND METHODS…………………………………………… 40
3.1. Materials ........................................................................................................... 40
Preparation of spice extracts…………………………………………………..40
Preparation and storage of cooked ground beef and pork patties…………….41
3.4 Proximate analysis ……………………………………………………….. . . 42
Moisture content42
Crude protein content43
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
Digestion and analysis for minerals …………………………………………..45
Determination of vitamin composition ……………………………………….47
Determination of ascorbic acid (Vitamin C) content ……………………...47
Determination of niacin content …………………………………………....47
Determination of riboflavin content ………………………………………..48
Determination of thiamin content …………………………………………..49
Determinations of phytochemical composition ……………………………49
Determination of total phenol ………………………………………………49
Determination of total flavonoids ……………………………………………50
Determination of condensed tannin content ………………………………….50
Determination of total anthocyanin content …………………………………51
Determination of carotenoid content ………………………………………….51
Determination of alkaloid content …………………………………………...52
Determination of phytate content ……………………………………………52
Determination of oxalate content …………………………………………….53
Saponin content determination ………………………………………………53
Determination of Antioxidant Properties of Spices …………………………..54
Determination of free radical scavenging activity54
Measurement of reducing power of the crude extracts of spices …………54
Determination of antioxidant activity of crude extracts of the spices by the Ferric thiocyanate (CTC) method ………………………………………..55
Determination of antimicrobial properties of the spices …………………55
minimum inhibitory concentration (MIC) determination ………………....55
Preparation and inoculation of substrates ………………………………..56
Bacterial strains and preparation of inoculants…………………………...57
Monitoring of survival and growth of pathogen population …………….57
Determination of Thiobarbituric acid (TBA) reactive substances in minced
meat patties during storage 58
Experimental design59
Statistical analysis59
CHAPTER FOUR
RESULTS AND DISCUSSIONS ………………………………………..60
Proximate composition and energy value of spices ………………………….60
Mineral composition of the spices …………………………………………….64
Vitamin content of the spices ………………………………………………69
Yield of crude extracts of spices ………………………………………………..73
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
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
Estimation of reducing power of spices……………………………………98
Reducing power of Ocimum viride ………………………………………………98
Reducing power of of Monodora myristica ……………………………………101
Reducing power of Monodora trifolia103
Reducing power of Tetrapleura tetrapetra ……………………………………105
Comparative effect of extracting solvents on reducing power of the four spices107
Inhibition of Linoleic acid peroxidation ………………………………… .109
Scavenging of 1,1 – diphenyl-2-picryl hydrazyl radical (DPPH) by the spices111
DPPH radical scavenging activity of Ocimum viride …………………………114
DPPH radical scavenging activity of Monodera myristica ……………………116
DPPH radical scavenging activity of Monodora tenuifolia…………………118
DPPH Radical scavenging activity of Tetrapleura tetrapetra ………………120
Comparison of DPPH radical scavenging activities of solvent extract of spices…………………..122
Inhibition of lipid peroxidation in cooked, ground meat patties by spices
during storage 124
Comparison of Mean TBA values of meat patties treated with spices
during storage ……………………………………………………………… 126
Mean TBA values for Ocimim viride ……………………………………..126
Mean TBA values for Monodora myristica ……………………………….129
Mean TBA values for Monodora tenuifolia…………………………………131
Mean TBA values for Tetrapleura tetropetra134
Antimicrobial activities of the spices against some selected food pathogens138
Sensitivity of the three bacteria toward inhibitory activity of aqueous
and ethanol extracts of the spices .......................................................... …. 138
Growth of pathogens in control substrates …………………………………141
Antibacterial activities of of Monodora myristica against food borne
Pathogens 142
Antibacterial activities of Monodora tenuifolia against food borne
pathogens 148
Antibacterial activities of Ocimum viride against food borne pathogens154
Antibacterial activities of Tetrapleura tetrapetra against food borne pathogens …… 160
Phytochemical composition of cooked spice-treated food extracts168
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS 173
CONCLUSION173
RECOMMENDATIONS 175
References 176
Appendices 193
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
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 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
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
CHAPTER ONE
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
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).
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
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.
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.
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.
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.
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.
.