CHEMICAL COMPOSITION AND THE EFFECT OF EXTRACTION SOLVENTS ON THE ANTIOXIDANT CONTENTS OF VITEX DONIANA (UCHAKURU) LEAVES

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ABSTRACT

The chemical composition and the effect of different extraction solvents (water, acetone, ethanol and water-acetone (50:50)) on the leaf extracts’ antioxidant and antioxidant activities were also analyzed. The results revealed that the proximate composition had 4.5, 16.21, 11.09, 4.07, 9.17, 54.89 and 321.09 Kcal for moisture content, crude protein, crude fibre, fats, ash, carbohydrates and energy value respectively. Vitamins A, B1, B2, B3, B6, C and E were found to be present in the leaves with vitamin C had the highest cponcentration (110.14 mg/100g). Mineral content showed 59.73, 8.71, 17.55, 45.05, 116.83, 18.63 and 3.93 mg/100g for calcium, sodium, magnesium, phosphorus, potassium, iron and zinc respectively. Antinutrients values of 3.74, 0.92, 5.63, 8.92, 0.67 and 6.33 mg/100g for alkaloid, tannin, saponin, phytate, oxalate and glycoside respectively. The results showed that V. doniana leaves are rich in flavonoids and phenolic constituents. The highest total phenolic content of 451.03 mgGAE/g was obtained in water extract and highest total flavonoid extraction of 302.92 mgQE/g in ethanol extract. The antioxidant activities (FRAP and ABTS) were strongest in ethanol extract, (water 0.94, acetone 1.06, water-acetone (50:50) 0.77, ethanol 1.40), (water 61.03, acetone 32.78, water-acetone (50:50) 40.72, ethanol 66.31) respectively while the DPPH activities was strongest in water extract. (water 88.29, acetone 50.17, water-acetone (50:50) 51.13, ethanol 54.07). Utilization of Vitex doniana (uchakuru) leaves should be encouraged for use As food for its nutrients and antioxidant properties to enhances the nutritional and health status of the consumers. 





TABLE OF CONTENTS

Title                                                              i
Declaration                                                        ii
Certification                                                       iii
Dedication                                                        iv
Acknowledgement                                                  v
Table of Contents                                                   vi
List of Tables                                                      vii
List of Figures                                                     viii
List of Plates                                                       ix
List of Appendix                                                   x
Abstract                                                          xi

CHAPTER 1: INTRODUCTION
1.1 Background of the study                                          1
1.2 Statement of problem                                             2
1.3 Justification of the study                                          2
1.4 Objectives of the study                                           3

CHAPTER 2: LITERATURE REVIEW
2.1Uchakuru (Vitex doniana)                                         4
2.2 Uchakuru as an underutilized indigenious vegetable          5
2.3 Nutritional benefits of uchakuru leaves                              8
2.4 Medicinal and health benefits of uchakuru leaves         9
2.5 Extracts of V. doniana leaf and extracting liquids          10
2.6 Anti-nutritional properties of uchakuru leaves                         11
2.7 Antioxidant and phytochemical properties of V. doniana leaves           12                                 
2.8 Relationship between antinutrients and antioxidants           15
2.8.1 Saponins                                                     15
2.8.2 Phytate                                                      16

CHAPTER 3: MATERIALS AND METHODS 
3.1 Source of materials                                              17
3.2Sample preparation                                              17
3.2.1 Preparation of Vitex doniana((uchakuru) leaf extract        17
3.3 Proximate analysis                                              20
3.3.1 Moisture determination                                         20
3.3.2 Determination of crude protein                                   20
3.3.3 Crude fiber determination                                       21
3.3.4 Ash content determination                                      22
3.3.5 Determination of fat content                                     22
3.3.6 Total carbohydrate determination                                 22
3.3.7Determination of caloric value                                    23
3.4Vitamin analysis                                                23
3.4.1 Determinationvitamin A (retinol)concentration            24
3.4.2 Determination of vitamin B1 (thiamine)concentration      24
3.4.3 Determinationvitamin B2 (riboflavin)concentration         24
3.4.4 Determination of vitamin B3 (niacin)                             25
3.4.5Determination of vitamin B6(pyridoxine )                          25
3.4.6Determination of vitamin C (ascorbic acid) concentration  25
3.4.7Determination of vitamin E (α -tocopharol) concentration   26
3.5Minerals analysis                                               26
3.5.1 Calcium determination                                        26  
3.5.2Sodium determination                                         27
3.5.3Magnesium determination                                      27
3.5.4Determination of phosphorus                                            28
3.5.5Determination of potassium                                              28
3.5.6Iron determination                                            29
3.5.7Determination of zinc                                                    29       3.6  Antinutrients  analysis                                        30
3.6.1 Alkaloid determination                                         30
3.6.2 Tannin determination                                          30
3.6.3 Saponin determination                                         31
3.6.4 Phytate determination                                          31
3.6.5 Oxalate determination                                         31
3.6.6 Glycoside determination                                       32               
3.7 Antioxidant analysis                                            32
3.7.1 Total phenolic determination                                    33
3.7.2 Total flavonoids content                                        33
3.7.3 Antioxidant activity determinations                               33
3.7.3.1 DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging assay      33
3.7.3.2 Ferric ion reducing antioxidant power (FRAP) assay                34
3.7.3.3 ABTS (2,2’-azinibis-(3-ethyl benzothiazoline-6sulphonic acid) assay   34
3.8 Experimental design                                             35
3.9 Statistical analysis                                               35

CHAPTER 4: RESULTS AND DISCUSSIONS
4.1 Proximate composition of uchakuru (V. doniana) leaves (dry matter basis)  36                           
4.2 Vitamin content of uchakuru leaves (dry matter basis)                  39
4.3 Mineral content of uchakuru leaves (dry basis)                        41
4.4 Antinutritional composition of uchakuru leaves (dry basis)              45                                                 
4.5 Antioxidants in uchakuru leaf extract                               49
4.6 Antioxidant activity of uchakuru leaf extracts                        51

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion                                                    56
5.2 Recommendation                                               56
References                                                       57
Appendices                                                      63






LIST OF TABLES

Table 4.1: Proximate composition of uchakuru leaves          37

Table 4.2: Vitamin content of uchakuru leaves                           40

Table 4.3: Mineral content of uchakuru leaves                           42

Table 4.4: Antinutritional / phytochemical composition of uchakuru leaves    47                                              




LIST OF FIGURES

Figure 1: Flow chart for preparation of uchakuru(Vitex doninana) leaves extract with modifications to method described by Yakubu et al. (2016a)      19

Figure 2: Graphical representation of uchakuru leaf extract antioxidants and composition in mgQE/g and mgGAE              50 

Figure 3: Graphical representation of uchakuru leaf extracts % DPPH Inhibition at various concentrations compared to vitamin C as standard         52       
              
Figure 4: Graphical representation of uchakuru leaf extracts FRAP assay at various concentrations compared to Trolox       53

Figure 5: Graphical representation of uchakuru leaf extracts % ABTS Inhibition at various concentrations compared to trolox  54






LIST OF PLATES

Plate 1: Pictures of uchakuru (Vitex doniana) leaves         18






LIST OF APPENDIX

Appendix 1: Pictures of project student in the laboratory work                63

Appendix 2: Result of % DPPH inhibition of uchakuru leaf extract            64

Appendix 3: Result of FRAP assay of uchakuru leaf extract                  65

Appendix 4: Result of % ABTS  inhibition of uchakuru leaf extracts          66






CHAPTER 1
INTRODUCTION

1.1 BACKGROUND OF THE STUDY.
Vegetables, especially leafy vegetables are important source of vitamins, minerals, fibres, and some essential amino acids. They have high content of water and abundance of cellulose. The cellulose is in a form which although not digested, serves a useful purpose in the intestine as roughages, thus promoting normal elimination of waste products (Osum et al., 2013).

Vitex doniana(uchakuru) is an indigenous fruit or leafy vegetable in Africa (Dako et al., 2014). It is readily recognized by its long-stalked gabbros leaves with the leaflets usually rounded at the apex, though sometimes they may be unrented or occasionally have very short triangular tip and the fruits are edible (Osum et al., 2013). V. doniana is one of the most consumed leafy vegetables collected from the wild (Achigan-Dako et al., 2011). The dried leaves are used to prepare sauces. In folk medicine, the leaves are employed in the treatment of stomach pains and in case of frequent stooling. They are usually chewed, extracted with water or used to prepare yam porridge. It is traditionally known that the efficiency or potency of V. doniana leaf extract increases with the intensity of the bitterness, it’s water extract is of primary interest. In addition, the plant is used in the treatment of mouth ulcers and sores. In this situation, the leaves are washed and chewed after which they are swallowed. The young leaves are used for stock in the Northern part of the country and also as pot herbs (Osum et al., 2013).

1.2 STATEMENT OF PROBLEM
Nigeria is blessed with different varieties of fruits and vegetables like vitex doniana (uchakuru), but most of them are underutilized due to lack of adequate information on their nutritional and health benefits. Also, some vegetables (like Uchakuru) are very high in anti-nutritional factors (like tannins, saponnins, alkaloids, phenols, etc.) which are chemical substances that inhibit the proper absorption of nutrients (Arokiyaraj et al., 2009; Adejumo et al., 2013). Consumption of these vegetables without proper preparation can lead to malnutrition especially among the rural dwellers who consume Uchakuru without proper utilization of the nutrients (Enechi and Odonwodu, 2003; Soetan and Oyewole, 2009; Achigan-Dako et al., 2011; Okoye, 2015). 

1.3 JUSTIFICATION OF THE STUDY
Vegetables, especially leafy vegetables like Uchakuru are an important source of vitamins, minerals, fibre, and some essential amino acids (Osum et al., 2013), but most of these nutrients are not absorbed by the body due to presence of anti-nutritional factors (Soetan and Oyewole, 2009; Arokiyaraj et al., 2009; Adejumo et al., 2013). Nevertheless, some of the commonly considered anti-nutrient compounds like saponins and tannins are now being considered as potential antioxidants with health benefits and termed phytochemicals. Hence, depending on the consumer preferences retaining or eliminating these compounds could be facilitated (Awoyinka et al., 2016). Analysis of the chemical and antioxidant properties of vitex doniana (uchakuru) leaves extract will reveal its nutrients, anti-nutrients, antioxidants and health benefits and thereby encourage its proper utilization (Enechi and Odonwodu, 2003; Soetan and Oyewole, 2009; Adejumo et al., 2013).

1.4 OBJECTIVES OF THE STUDY
The main objective of this study was to obtain ‘chemical composition and the effect of extraction solvents on the antioxidant contents of vitex doniana (uchakuru) leaves obtained from Owelli-Court in Awgu Local Government Area of Enugu State.

The specific objectives were to:

i. Obtain and air dry Uchakuru leaves.

ii. Determine the proximate, vitamins, mineral and antinutrient composition of the dried leaves.

iii. Prepare water, acetone, ethanol and water-acetone (50:50) Uchakuru leaf extract, respectively.

iv. Determine the antioxidant and antioxidant activities of the leaf extracts.


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