C1 - Internal use A N I N V E S T I G A T I O N I N T O T H E C H E M I C A L C O M P O S I T I O N O F V A R I O U S S E E D O I L S F O R A P P L I C A T I O N I N C O S M E T I C P R O D U C T S Sabrina Tanita Khoosal 1385185 A dissertation submitted to the faculty of science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of master of science. University of the Witwatersrand Johannesburg 2021 i C1 - Internal use DECLARATION I hereby declare that this dissertation is a result of my own, unaided work except where acknowledged. It is being submitted in fulfilment of the requirements for the degree Master of Science in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination in any other University. _______________________________________ Sabrina Tanita Khoosal ____4th___________day of ______June_________2022________at_____Roodepoort_________ ii C1 - Internal use ABSTRACT Due to increased consumer awareness, there has been a strong shift in cosmetic product trends towards more natural and/ or greener alternatives in recent years. This is due to several factors such as cases of adverse effects caused by certain synthetic ingredients in products as well as the awareness of synergistic benefits of natural ingredients. Seed oils are one of the solutions being used to satisfy this newly found demand due to their chemical compositions that have cosmetic, nutritional and medicinal benefits (Antignac et al., 2011; Vermaak et al., 2011; Dangarembizi et al., 2015). Seed oils consist of essential and nonessential fatty acids, antioxidants, and vitamins, amongst other phytochemicals. This makes seed oils useful as emollients, active ingredients or as carriers for other active ingredients (Vermaak et al., 2011; Dangarembizi et al., 2015). The seed oils focused on in this study are from underutilised plants native to, or highly cultivated in Africa (Vermaak et al., 2011; Dangarembizi et al., 2015). They are namely Moringa oleifera (MO), Sclerocarya birrea (Marula), Citrullus lanatus (Kalahari melon), Schinziophyton rautanenii (Mongongo), Adansonia digitate L. (Baobab), and Cannabis sativa L. (Hemp). Other commercially available plant oils included for comparative purposes in the study include avocado oil, olive oil, jojoba seed oil, grapeseed oil and sunflower seed oil. Increasing the commercialisation of these underutilised oils will benefit rural communities involved in harvesting, encourage maintenance of the natural environment, provide more export products and alternative natural ingredients for the cosmetic industry, which will overall benefit our bioeconomy (Zimba et al., 2005; Juliani et al., 2007; Vermaak et al., 2011). Research is therefore needed to contribute to the database of information on these oils to further understand their potential in the natural-based cosmetic sector. To evaluate the economic importance and potential of these seed oils for cosmetic applications, in-depth profiling of the oils were conducted (Cheikhyoussef, 2018). The oil profiles include physico-chemical properties; namely acid value (AV), saponification value (SV), average molecular weight (AMW), ester value (EV), peroxide value (PV), iodine value (IV), specific gravity and the refractive index. Further characterisation of the oils involved Fourier Transform-Infrared Spectroscopy (FT-IR) analysis, antioxidizing abilities using Ultraviolet-visible spectroscopy (UV- iii C1 - Internal use Vis), chemical composition using Nuclear Magnetic Resonance (NMR) Spectroscopy, thermal behaviour using Differential scanning Calorimetry (DSC) as well the fatty acid methyl ester (FAME) profile using Gas Chromatography-Mass Spectrometry (GC-MS) as well as studying the effect of sunlight exposure on the various oils. AV is a reflection of the total acidity of the oil sample; therefore a lower value indicates less degradation and therefore higher quality of the oil and vice versa, the lowest AV amongst the oils of interest was obtained by Baobab oil (0.40 ± 0.01 mg KOH/g oil). Peroxide value (PV) is a chemical characteristic used to determine the extent of oxidative deterioration of the oil at the time of analysis, the lowest PV from the oils of interest belonged to Baobab oil. AV and PV both confirm the stability and high quality of Baobab making it very important to the cosmetic industry as products often have shelf-lives of 12 months. SV is a measure of the chain length of fatty acids present in the oil and is used to calculate an average molecular weight (AMW). The specific SV range optimal for soap making is 188 to 199 mg KOH/ 1g oil (Francis and Tahir, 2016). Therefore from the oils studied, Plush Organics Moringa and Hemp seed oil having SV of 189.23 ± 0.95 mg KOH /1g oil and 195,05 ± 0.56 mg KOH /1g oil respectively, would be most suitable for application in soap making. IV is related to the degree of unsaturation such that the higher the IV the more unsaturated the oil. The IV obtained for the extracted MO oils (65.93-74.99 g/100g) was lower than that obtained for Baobab seed oil (91.30 g/100g) and Kalahari melon seed oil (131.90 g/100g). Relative IV from 1H NMR data for Lefakong farming cold- pressed MO oil and Soxhlet extracted MO oil (68.54-69.49 g/100g) was lower than that obtained for Baobab seed oil (76.44 g/100g) and Kalahari melon seed oil (126.36 g/100g). From relative fatty acid percentages, the TUFA% results for the extracted MO oils (33.93-64.33%) is lower than the TUFA% obtained for Baobab seed oil (65.61%) and Kalahari melon seed oil (72.24%). Multiple parameters therefore confirm the result that Kalahari melon seed oil was the most unsaturated followed by Baobab seed oil and MO oil was the least unsaturated from this sample group. iv C1 - Internal use From the effect of sunlight exposure study there was a clear general trend amongst the oils as they deteriorated with time. Many of the selected oils lost their original colour and became pale to translucent; many also lost their original earthy and nutty smells and formed acidic and rancid odours which are clear signs of deterioration. Studies conducted on the thermal properties of selected oils using DSC showed multiple endothermic and exothermic peaks, indicating that there are multiple transition temperatures experienced by each oil due to their unique triacylglycerol profiles. There were no major differences between the Soxhlet extracted MO and cold-pressed MO, indicating that the extraction method used does not affect the thermal properties of oils. NMR results gave insight to the chemical composition of the oils and their degree of saturation which was confirmed with GC-MS fatty acid analysis. Fatty acid analysis indicated that Olive oil had the highest relative percentage of Oleic acid making it highly stable and able to withstand high temperatures hence its application in cooking. The highest relative percentage of Palmitic acid belonged to Grapeseed oil and the highest relative percentage of Linoleic acid belonged to Kalahari melon seed oil. This would indicate that Grapeseed oil and Kalahari melon seed oil are very valuable to skin formulations focussed on protecting the skin barrier from dying and damage. Radical scavenging activity of the oils indicate antioxidant content of the oil and therefore log term shelf- life of the oil. Results indicated that Kalahari melon seed oil and Mongongo had the highest antioxidant content which would make them ideal additives to antioxidant type cosmetic products. Trial application of oil enhanced cosmetic bases show a clear improvement in performance of products and an overall preferred richness in texture and feel. Overall, this study gave an in-depth insight into the chemistry behind the behaviour and properties of these various oils. This gives a deeper understanding of their potential application in cosmetic formulations. v C1 - Internal use PRESENTATION Participation in the National Young Chemist’s Symposium 2021 – MSc Flash Talk Presentation Title: An investigation into the chemical composition of various seed oils for application in cosmetic products vi C1 - Internal use In loving memory of my mother Meena Khoosal 1968-2015 All that I am, or hope to be, I owe to my angel mother -Abraham Lincoln vii C1 - Internal use ACKNOWLEDGEMENTS I would like to acknowledge The University of the Witwatersrand Postgraduate Merit Award (PMA) and the National Research Foundation (NRF) for the financial support, my supervisor Dr Heidi Richards and Co-supervisor Prof Luke Chimuka, for their guidance and supervision, Dr Yannik Nuapia and my other senior colleagues in the Environmental analytical chemistry research group for their help and support in the laboratory, Thapelo Mbhele for GC-MS training, Lefakong Farming for hosting us at their cold-pressing factory, my family and friends for their unwavering support, and lastly a special thank you to my father Kevin Khoosal and husband Mishaé Ramouthar for their all-round support and motivation. viii C1 - Internal use CONTENTS DECLARATION ........................................................................................................... i ABSTRACT ................................................................................................................. ii PRESENTATION ....................................................................................................... v ACKNOWLEDGEMENTS ......................................................................................... vii LIST OF FIGURES ..................................................................................................... xi LIST OF TABLES ..................................................................................................... xvi ABBREVIATIONS .................................................................................................. xviii CHAPTER 1: INTRODUCTION ................................................................................ 20 1.1 General Introduction ............................................................................................................. 20 CHAPTER 2: LITERATURE REVIEW ...................................................................... 23 2.1 Natural oils in cosmetics ....................................................................................................... 23 2.2 Profile and characteristics of the seed oils ............................................................................ 28 2.2.1 Moringa (Moringa oleifera) .......................................................................................... 28 2.2.2 Marula (Sclerocarya birrea) .......................................................................................... 30 2.2.3 Kalahari melon (Citrullus lanatus) ................................................................................. 33 2.2.4 Mongongo/ Manketti (Schinziophyton rautanenii) ...................................................... 35 2.2.5 Baobab (Adansonia digitate L.) ..................................................................................... 36 2.2.6 Hemp (Cannabis sativa L.)............................................................................................. 39 2.3 Extraction techniques ............................................................................................................ 41 2.3.1 Cold-press extraction .................................................................................................... 41 2.3.2 Soxhlet extraction ......................................................................................................... 42 2.3.3 Ultrasound-assisted extraction ..................................................................................... 44 2.4 Characterization and analytical techniques ........................................................................... 47 2.4.1 Gas Chromatography-Mass Spectrometry .................................................................... 47 2.4.1 Nuclear Magnetic Resonance ....................................................................................... 49 2.4.2 Ultraviolet-visible spectroscopy .................................................................................... 50 2.4.3 Differential scanning calorimetry .................................................................................. 52 2.4.4 Fourier transform- Infrared spectroscopy .................................................................... 54 CHAPTER 3: AIMS AND OBJECTIVES ................................................................... 56 3.1 Aims ......................................................................................................................................... 56 ix C1 - Internal use 3.2 Objectives ................................................................................................................................ 56 3.3 Motivation ............................................................................................................................. 57 CHAPTER 4: MATERIALS AND METHODS ........................................................... 57 4.1 Materials and reagents .......................................................................................................... 57 4.2 Extraction of Moringa oleifera seed oil ................................................................................ 58 4.2.1 Sample preparation ...................................................................................................... 58 4.2.2 Cold-press extraction .................................................................................................... 58 4.2.3 Soxhlet extraction ......................................................................................................... 61 4.2.4 Ultrasound-assisted extraction ..................................................................................... 62 4.2.5 Hexane extraction at ambient temperature (shaker method) ..................................... 62 4.2.6 Determination of percentage oil yield and efficiency................................................... 63 4.3 Determination of physico-chemical characteristics .............................................................. 63 4.3.1 Oil preparation .............................................................................................................. 63 4.3.2 Colour and state of oils ................................................................................................. 63 4.3.3 Determination of the acid value ................................................................................... 64 4.3.4 Determination of the saponification value and average molecular weight ................. 64 4.3.5 Determination of the ester value .................................................................................. 65 4.3.6 Determination of the iodine value ................................................................................ 65 4.3.7 Determination of the peroxide value ........................................................................... 66 4.3.8 Determination of density and specific gravity .............................................................. 67 4.3.9 Determination of the refractive index .......................................................................... 67 4.4 Determination of thermal properties ..................................................................................... 68 4.5 Fourier Transform-Infrared Spectroscopy analysis .............................................................. 68 4.6 Nuclear Magnetic Resonance (NMR) spectroscopy ............................................................. 69 4.7 Determination of fatty acid content ...................................................................................... 70 4.8 Effect of sunlight exposure ................................................................................................... 72 4.9 Determination of radical scavenging activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical .................................................................................................................................. 73 4.10 Statistical methods and analysis ............................................................................................ 73 4.11 Trial formulation application of cosmetic products .............................................................. 74 CHAPTER 5: RESULTS AND DISCUSSION ........................................................... 74 5.1 Moringa oleifera oil extraction yield and efficiency percentages......................................... 74 5.2 Physico-chemical characteristics of the various oils ............................................................. 76 5.2.1.1 Acid value ...................................................................................................................... 76 5.2.2 Saponification value and Average molecular weight .................................................... 77 5.2.3 Ester value ..................................................................................................................... 78 x C1 - Internal use 5.2.4 Iodine value ................................................................................................................... 78 5.2.5 Peroxide value ............................................................................................................... 80 5.2.6 Density and specific gravity .......................................................................................... 80 5.2.7 Refractive index ............................................................................................................ 81 5.3 Thermal properties of selected oils ....................................................................................... 85 5.4 Nuclear Magnetic Resonance (NMR) spectroscopy of selected oils .................................... 88 5.5 Fourier Transform-Infrared Spectroscopy analysis of the various oils ................................. 91 5.6 The relative fatty acid content of the various oils ................................................................. 93 5.7 The effect of sunlight exposure on selected oils ................................................................. 101 5.8 Radical scavenging activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical of the various oils ...................................................................................................................................... 107 5.9 Statistical analysis of quantitative parameters .................................................................... 111 5.10 Trial application of cosmetic products ................................................................................ 113 CHAPTER 6: CONCLUSION ................................................................................. 115 CHAPTER 7: RECOMMENDATIONS .................................................................... 117 REFERENCES ....................................................................................................... 118 APPENDICES ........................................................................................................ 140 APPENDIX A: DSC thermal profiles ............................................................................................. 140 APPENDIX B: NMR Spectra ......................................................................................................... 142 APPENDIX C: FT- IR Spectra ....................................................................................................... 146 APPENDIX D: Gas chromatograms for fatty acid profile.............................................................. 158 APPENDIX E : Cosmetic product questionnaire ........................................................................... 177 xi C1 - Internal use LIST OF FIGURES Figure 1: Chemical structures of important fatty acids (Vermaak et al., 2011) ......... 26 Figure 2: Chemical structures of important phytosterols(Vermaak et al., 2011) ....... 27 Figure 3: Chemical structures of tocopherols (Constantinou, Papas and Constantinou, 2008) ................................................................................................. 27 Figure 4: The pods, seeds and kernels of Moringa oleifera(Noh, 2018) ................... 29 Figure 5: Sclerocarya birrea (Marula) (Hetta, 2016) ................................................. 32 Figure 6: Citrullus lanatus (Kalahari melon) (Hetta, 2016) ........................................ 34 Figure 7: Schinziophyton rautaneii (Mongongo) (Hetta, 2016) ................................. 36 Figure 8: Adansonia digitatal (Baobab)(Hetta, 2016) ............................................... 38 Figure 9: Hemp seeds (left), a young hemp plant (centre), and a mature flowering hemp plant (Smith, 2018) ......................................................................................... 40 Figure 10: Modified illustration of cold-pressing (Vaughn et al., 2018) ..................... 42 Figure 11: Illustration of Soxhlet extraction apparatus (Bhutada et al., 2016) .......... 44 Figure 12: Schematic illustration of an ultrasonic bath and ultrasonic probe(Behzadnia, Moosavi-Nasab and Tiwari, 2019) .............................................. 45 Figure 13: Schematic plot of the main components of the GC–MS instrument (Fullan, 2015) ........................................................................................................................ 48 Figure 14: Schematic diagram of an NMR spectrometer(Rankin et al., 2014) ......... 50 Figure 15: Schematic diagram of a UV-Vis spectrometer(Sobarwiki, 2013) ............. 52 Figure 16: Schematic diagram of DSC(Bibi et al., 2015) .......................................... 54 Figure 17: Schematic diagram of FT-IR(Bibi et al., 2015) ........................................ 56 Figure 18: Illustration of the MO sample preparation steps from A- Whole seed, to B- De-shelled kernel and lastly C-Kernel/seed powder ................................................. 58 Figure 19: Illustrations of the MO oil Cold-pressing process (A-H) ........................... 61 Figure 20: Images of Soxhlet extraction process where A= Soxhlet tube set up and B= Rotary evaporator set up .................................................................................... 61 xii C1 - Internal use Figure 21: Illustration of the DSC instrument used in this study ............................... 68 Figure 22: Illustration of the FT-IR spectrophotometer set-up .................................. 69 Figure 23: Illustration of the NMR set-up .................................................................. 70 Figure 24: Illustration of the GC-MS set-up .............................................................. 72 Figure 25: Illustration of the selected oils at the start of the sunlight exposure study (14/12/2020). Where 1= Jojoba seed oil. 2= Grapeseed oil, 3= Avocado oil, 4= Sunflower oil, 5= Sunflower oil, 6= Lefakong Farming cold-press MO oil, 7= Bright mountains MO oil, 8= Marula Seed Oil, 9= Mongongo Seed oil, 10= Kalahari Melon Seed Oil, 11= Baobab Seed Oil, and 12= Hemp seed oil ......................................... 73 Figure 26: DSC cooling profile of various seed oils .................................................. 87 Figure 27: DSC heating profile of various seed oils.................................................. 87 Figure 28: Overlapped FT-IR Spectra of extracted MO oil ....................................... 93 Figure 29: GC-MS chromatogram of Shaker extracted Moringa seed oil ................. 97 Figure 30: Images of the selected oils during the sunlight exposure study A= 14/12/2020, B= 14/05/2021 and C=14/08/2021. Oils were positioned as follows: 1= Jojoba seed oil. 2= Grapeseed oil, 3= Avocado oil, 4= Sunflower oil, 5= Sunflower oil, 6= Lefakong Farming cold-press MO oil, 7= Bright mountains MO oil, 8= Marula Seed Oil, 9= Mongongo Seed oil, 10= Kalahari Melon Seed Oil, 11= Baobab Seed Oil, and 12= Hemp seed oil .................................................................................... 103 Figure 31: Covariance matrix of quantitative results. Where AV = Acid value, SV = Saponification value, AMW = Average molecular weight, EV = Ester value, IV = Iodine value, PV = Peroxide value, D = Density, SG = Specific gravity, RI = Refractive index, and RSA = Radical scavenging ability ........................................ 113 Figure 32: Illustration of cosmetic product samples ............................................... 114 Figure 33: Complete DSC thermal profile of baobab oil ......................................... 140 Figure 34: Complete DSC thermal profile of cold-pressed MO oil .......................... 140 Figure 35: Complete thermal profile of Kalahari melon seed oil ............................. 141 Figure 36: Complete thermal profile of Soxhlet extracted MO oil ........................... 141 xiii C1 - Internal use Figure 37: 1H NMR (500 MHz, CDCl3) spectrum of Lefakong farming cold-pressed MO seed oil ............................................................................................................ 142 Figure 38: 13C NMR (500 MHz, CDCl3) spectrum of Lefakong farming cold-pressed MO seed oil ............................................................................................................ 142 Figure 39: 1H NMR (500 MHz, CDCl3) spectrum of Soxhlet extracted MO seed oil 143 Figure 40: 13C NMR (500 MHz, CDCl3) spectrum of Soxhlet extracted MO seed oil ............................................................................................................................... 143 Figure 41: 1H NMR (500 MHz, CDCl3) spectrum of Kalahari melon seed oil .......... 144 Figure 42: 13C NMR (500 MHz, CDCl3) spectrum of Kalahari melon MO seed oil .. 144 Figure 43: 1H NMR (500 MHz, CDCl3) spectrum Baobab seed oil ......................... 145 Figure 44: 13C NMR (500 MHz, CDCl3) spectrum of Baobab seed oil .................... 145 Figure 45: FT-IR spectra of Ultrasound-assisted extracted MO seed oil ................ 146 Figure 46: FT-IR spectra of Lefakong farming cold-press MO seed oil .................. 146 Figure 47: FT-IR spectra of Soxhlet extracted MO seed oil .................................... 147 Figure 48: FT-IR spectra of Shaker method extracted MO seed oil ....................... 147 Figure 49: FT-IR spectra of Moyo moringa SA seed oil .......................................... 148 Figure 50: FT-IR spectra of Leaf- Moringa seed oil ................................................ 148 Figure 51: FT-IR spectra of Essentially Natural Moringa oil ................................... 149 Figure 52: FT-IR spectra of Moringa 5000 Pure Moringa Ben oil ........................... 149 Figure 53: FT-IR spectra of Bright mountain - Moringa Seed Oil ........................... 150 Figure 54: FT-IR spectra of Moriveda moringa basic oil ......................................... 150 Figure 55: FT-IR spectra of Plush Organics Moringa Oil ........................................ 151 Figure 56: FT-IR spectra of Tree of life Pure organic moringa oil ........................... 151 Figure 57: FT-IR spectra of Moringa world MO oil ................................................. 152 Figure 58: FT-IR spectra of Baobab seed oil .......................................................... 152 Figure 59: FT-IR spectra of Kalahari Melon seed oil .............................................. 153 Figure 60: FT-IR spectra of Marula seed oil ........................................................... 153 xiv C1 - Internal use Figure 61: FT-IR spectra of Mongongo seed oil ..................................................... 154 Figure 62: FT-IR spectra of Hemp seed oil ............................................................ 154 Figure 63: FT-IR spectra of Avocado seed oil ........................................................ 155 Figure 64: FT-IR spectra of Grapeseed oil ............................................................. 155 Figure 65: FT-IR spectra of Jojoba seed oil ........................................................... 156 Figure 66: FT-IR spectra of Sunflower seed oil ...................................................... 156 Figure 67: FT-IR spectra of Olive oil....................................................................... 157 Figure 68: GC-MS chromatogram of Supelco 37 component FAME mix reference 160 Figure 69: Mass spectra of major FAMES oleic acid, palmitic acid and linoleic acid ............................................................................................................................... 160 Figure 70: Gas chromatogram of Mongongo seed oil ............................................ 161 Figure 71: Gas chromatogram of Olive oil .............................................................. 161 Figure 72: Gas chromatogram of Plush organics Moringa oil ................................. 162 Figure 73: Gas chromatogram of Baobab seed oil ................................................. 162 Figure 74: Gas chromatogram of Kalahari melon seed oil ..................................... 163 Figure 75: Gas chromatogram of Bright mountain Moringa seed oil ...................... 163 Figure 76: Gas chromatogram of Moyo moringa SA oil.......................................... 164 Figure 77: Gas chromatogram of Sunflower seed oil ............................................. 164 Figure 78: Gas chromatogram of Moringa world oil ............................................... 165 Figure 79: Gas chromatogram of Karibaa - Moriveda moringa basic oil................. 165 Figure 80: Gas chromatogram of Essentially Natural Moringa oil .......................... 166 Figure 81: Gas chromatogram of Grapeseed oil .................................................... 166 Figure 82: Gas chromatogram of Avocado oil ........................................................ 167 Figure 83: Gas chromatogram of Jojoba seed oil ................................................... 167 Figure 84: Gas chromatogram of Tree of life Pure organic moringa oil .................. 168 Figure 85: Gas chromatogram of Moringa 5000 Pure Moringa Ben oil .................. 168 xv C1 - Internal use Figure 86: Gas chromatogram of Leaf- Moringa seed oil ....................................... 169 Figure 87: Gas chromatogram of Hemp seed oil .................................................... 169 Figure 88: Gas chromatogram of Marula seed oil .................................................. 170 Figure 89: Gas chromatogram of Lefakong Cold-pressed MO oil .......................... 170 Figure 90: Gas chromatogram of Soxhlet extracted MO oil .................................... 171 Figure 91: Gas chromatogram of Ultrasound extracted MO oil .............................. 171 xvi C1 - Internal use LIST OF TABLES Table 1: Major physico-chemical characteristics literature values of MO seed oil (Cold-pressed) ......................................................................................................... 30 Table 2: Major physico-chemical characteristics literature values of Marula seed oil (Cold-pressed) ......................................................................................................... 32 Table 3: Major physico-chemical characteristics literature values of Kalahari melon seed oil (Cold-pressed) ............................................................................................ 34 Table 4: Major physico-chemical characteristics literature values of Mongongo seed oil (Cold-pressed) ..................................................................................................... 36 Table 5: Major physico-chemical characteristics literature values of Baobab seed oil ................................................................................................................................. 38 Table 6: Major physico-chemical characteristics literature values of Hemp seed oil (Cold-pressed) ......................................................................................................... 41 Table 7: Average literature values for MO oil extraction yields and efficiency .......... 45 Table 8: MO oil extraction yield and extraction efficiency percentages .................... 76 Table 9: Physico-chemical characteristics of the various oils ................................... 83 Table 10: Transition temperatures during cooling processes of various seed oils ... 88 Table 11: Transition temperatures during heating processes of various seed oils ... 88 Table 12: Chemical shifts and assignments of main resonances in ppm (δ) of the 13C NMR spectra of selected seed oils ................................................................... 90 Table 13: Chemical shifts and assignments of main resonances in ppm (δ) of the 1H NMR spectra of selected seed oils ........................................................................... 90 Table 14: Integrals of the 1H NMR main resonance groups for the selected oil samples (% from the total area of the signals) ......................................................... 91 Table 15: Common FT-IR signals for the various seed oils (Appendix C Figure 45- 67) ............................................................................................................................ 92 Table 16: Relative fatty acid composition of the various oils .................................... 98 Table 17: Physical effect of sunlight exposure summary on various oils ................ 104 xvii C1 - Internal use Table 18: % Inhibition results for various oils ......................................................... 110 Table 19: Supelco 37 component FAME Mix FAME analysis data used for quantitation & identification of the unknown peaks in the oil samples. ................... 158 Table 20: Additional relative fatty acid content of various oils ................................ 172 xviii C1 - Internal use ABBREVIATIONS ANOVA Analysis of Variance AOCS American Oil Chemists’ Society AMW Average molecular weight AV Acid value 13C NMR Carbon 13 Nuclear Magnetic Resonance DPPH DI 1,1-diphenyl-2-picrylhydrazil Deionised EV Ester value FA Fatty acid FAMEs Fatty acid methyl esters FFA Free fatty acid 1H NMR Proton Nuclear Magnetic Resonance GC-MS Gas Chromatography-Mass Spectrometry IV MO MUFA Iodine value Moringa Oleifera Monounsaturated fatty acid nd not detected xix C1 - Internal use PUFA Polyunsaturated fatty acid PV Peroxide value R2 Coeffcient of determination RI SAFA Refractive index Saturated fatty acid SG Specific gravity SV Saponification value TAG Triacylglycerides TFA TUFA Trans Fatty Acids Total Unsaturated Fatty Acids 20 C1 - Internal use CHAPTER 1: INTRODUCTION 1.1 General Introduction Seed oils were historically used for cosmetic purposes, however, during the last century, convenient synthetic or petroleum-based substitutes, such as mineral oil and silicones, have replaced them (Vermaak et al., 2011). Synthetic based cosmetics have the advantage of lower cost and a longer shelf-life but have the disadvantages of at times causing adverse side effects, allergic reactions, and producing waste that is harmful to the environment. Whereas natural-based cosmetics have the advantage of being more environmentally friendly and causing fewer side effects, but have the disadvantage of costing more and having a shorter shelf-life (Chen, 2009). Due to the potential adverse effects of the synthetic materials, there is currently a growing trend to replace them with natural oils, butters and waxes in the cosmetic and pharmaceutical industries (Shackleton et al., 2006; Vermaak et al., 2011; Hetta, 2016). In modern years there has been an increase in consumers awareness towards ingredients used and the validity of performance claims made on cosmetic products. This has led to a higher global demand for more high- quality natural-based cosmetic products (Vermaak et al., 2011; Hetta, 2016). Seed oils are one of the solutions being used to satisfy this problem due to their chemical composition that have cosmetic, nutritional and medicinal benefits (Antignac et al., 2011; Vermaak et al., 2011; Dangarembizi et al., 2015). The seed oils are used as emollients, active ingredients or as carriers for other active ingredients (Vermaak et al., 2011; Dangarembizi et al., 2015). The seed oils focused on in this study include Moringa, Marula, Kalahari melon, Mongongo, Baobab, and Hemp seed oil. These oils are native to, or highly cultivated in Africa, which has rich biodiversity (Vermaak et al., 2011; Dangarembizi et al., 2015). These plants are used by locals for food, medicine, cosmetics, fuel etc. (Zimba et 21 C1 - Internal use al. 2005; Vermaak et al., 2011). Increasing commercialisation of these oils will benefit rural communities involved in harvesting, encourage maintenance of the natural environment, provide more export products and alternative natural ingredients for the cosmetic industry which will overall benefit our bioeconomy (Zimba et al. 2005; Juliani et al., 2007; Vermaak et al., 2011). These non-traditional seed oil sources, if managed correctly, can satisfy the ever-growing demand for this important renewable commodity by supplementing the worlds current vegetable oil supplies (Mitei et al., 2008; Janaki, 2015). Moringa oleifera (MO) is currently one of the most attractive plants under study in many disciplines due to its versatility and high nutritional value (Mehta et al., 2011). In a study by Özcan and co-workers, it was concluded that variations in MO oils nutritional value are likely due to differences in plant species, locations, climatic factors, harvesting times, cultivation conditions and extraction processing. Thus, to determine the best extraction method of MO oil for cosmetic purposes, a comparative study had to be conducted on the same MO source using different extraction methods. Cold-press extraction is one of the most attractive green extraction methods compared to traditional Soxhlet extraction. Previous extraction of MO oil have shown that in general, fatty acids, tocopherol and sterol contents recovered using cold-press extraction were higher in comparison to those obtained using Soxhlet extraction but resulted in lower yields (Özcan et al., 2019). To assess the economic importance and potential of these seed oils for cosmetic applications, an in-depth profiling of the oils needed be conducted (Cheikhyoussef, 2018). The profile of oils mainly includes physico-chemical properties; namely acid value (AV), ester value (EV), saponification value (SV), average molecular weight (AMW), iodine value (IV), peroxide value (PV), density, specific gravity and the refractive index. The triglycerol analysis using nuclear magnetic resonance spectroscopy 22 C1 - Internal use (NMR), antioxidizing abilities using ultraviolet-visible spectroscopy (UV- Vis) as well as the tocopherol, major sterol content and most importantly the fatty acid profile using gas chromatography-mass spectrometry (GC- MS). The increase in demand for natural-based cosmetics is currently not being met by industry which results in high prices for the limited range available. An increase in the availability of natural-based cosmetics will result in lower prices allowing more people to access this alternative. Further research needs to be done on the potential of renewable seed oils to attract industries to grow the natural-based cosmetic sector in Africa and benefit our bioeconomy (Górnaś and Rudzińska, 2016). Although researchers have already published information on numerous oils covered in this study, it is often difficult to compare data from various sources due to the high variability between techniques used for analysis (Tan and Che Man, 2000). Hence the need for this broad study that looked at MO oil extracted by four different methods, as well as profiling these oils alongside underutilised African oils in comparison to common vegetable oils such as olive and sunflower oil. 23 C1 - Internal use CHAPTER 2: LITERATURE REVIEW 2.1 Natural oils in cosmetics Natural oils are high in fatty acids, which are carboxylic acids with a long aliphatic chain. When three fatty acids are combined with glycerol they form triglycerides which are a component of fat (Leite and Maia Campos, 2018). Fats with a high unsaturated fatty acid content are usually in liquid form whereas those with a high percentage of saturated fatty acids are predominantly in solid form at room temperature. Seed oils are largely made up of unsaturated fatty acids but may contain some saturated fatty acids (SAFAs) such as palmitic, myristic and stearic acid. Unsaturated fats are categorised as monounsaturated fats, made up of omega 9 fatty acids or monounsaturated fatty acids (MUFAs), and polyunsaturated fats, which are made up of either omega 6 fatty acids or omega 3 fatty acids also known as n6 polyunsaturated fatty acids (PUFAs) or n3 PUFA respectively. The most important fatty acids in cosmetic formulations include palmitic (SAFA), oleic (MUFA) and linoleic (PUFA) acid (Lautenschläger, 2003). Palmitic acid is an important component of the natural skin barrier, it is used as a skin permeation enhancer for co- administered molecules (Vermaak et al., 2011; Hetta, 2016). Oleic acid enhances antioxidative ability and is effective as an absorption enhancer. Linoleic acid is not produced in the human body naturally and is, therefore, the most frequently used in cosmetic formulations. A deficiency of this fatty acid leads to water loss from the skin's epidermis resulting in dryness and scaling, it may also cause hair loss and cracking of nails (Hetta, 2016). Linoleic acid in cosmetic formulations is used to prevent disorders of the skin barrier, lower trans epidermal water loss which therefore moisturises the skin, treat sunburns and regenerate epithelial tissue as it is a membrane-forming phospholipid (Lautenschläger, 2003; Hetta, 2016).In general, a deficiency of these important fatty acids from the skin may lead to erythema, inflammation, poor wound healing and dryness (Brenner, 24 C1 - Internal use 2004). These fatty acids are known for their properties in isolation, it is believed that using the crude natural oil sources as a carrier for other active ingredients may have valuable synergistic effects (Vermaak et al., 2011; Hetta, 2016). Figure 1 below shows some of the major fatty acid’s chemical structures. Oils that contain natural antioxidants such as vitamin E have a substantially longer shelf life (Lautenschläger, 2003). An antioxidant is a preservative that reduces the rate of oxidation in oils which is a chemical process that occurs on exposure to oxygen (Muda et al.,2017). Vitamin E includes eight types of molecules, four tocotrienols and four tocopherols, which exhibit vitamin E activity. The purpose of vitamin E in cosmetic formulations is therefore to extend its shelf-life and to stop chain propagation in lipid peroxidation by scavenging lipid peroxyl radicals which defends the cell membrane against destruction. This in turn prevents the signs of photoaging (Muda et al.,2017). Figure 3 below shows the chemical structures of the four tocopherols. Important plant sterols or phytosterols found in the unsaponifiable fraction of seed oils include stigmasterol, β-sitosterol and campesterol. Sterols molecular structures are made up of rings, some containing unsaturated bonds, as well as branched hydrocarbon tails. When absorbed from the diet, phytosterols reduce the uptake of cholesterol in the body and are transferred to the skin where they make up an important component of the skins surface lipids (Puglia and Bonina, 2008). It is reported that phytosterols repair skin damage by having anti-inflammatory and antipruritic properties. Sitosterol has emulsifying effects due to its hydroxyl group that is attached to four lipophilic carbon rings. Phytosterols have been used in creams, conditioners and lipsticks due to their valuable properties (Svensson and Brinck, 2003; Puglia and Bonina, 2008). Figure 2 below shows the chemical structures of the major phytosterols of interest in this study. 25 C1 - Internal use Due to factors such as environmental elements, ultraviolet radiation, air pollution and natural ageing processes; the barrier properties of the skin and hair may be altered which results in damage. The skin is made up of three layers the deepest being the hypodermis, followed by the dermis above it and finally the top layer the epidermis. Skin damage can result in dry, flaky and rough skin that is easily irritated as well as stretch marks, wrinkles or thin and flaccid skin (Muda et al.,2017). Oils incorporated into cosmetic skin formulations such as moisturisers, therefore, protects the skin by reducing water loss. The hair shaft structure is made up of three layers, the innermost layer being the medulla, followed by the cortex and finally the cuticle which protects the two inner layers. In the case of damaged hair, the cuticle layer is not properly orientated in a flat and tightly overlapped configuration and therefore cannot protect the inner layers as effectively. Hair damage results in dry and dull hair that easily breaks (Muda et al.,2017). Oils incorporated into cosmetic hair formulations such as leave-in conditioners penetrate the hair shaft to enhance lubrication and therefore protect the hair from breakage (Gavazzoni Dias, 2015). In general, oils leave a transparent, water- repellent film on the surface of the skin and hair which reduces friction, adds shine and increases the softness of the skin and hair (Schueller and Romanowski, 2003; Muda, Aziz and Aziz, 2017). 26 C1 - Internal use Figure 1: Chemical structures of important fatty acids (Vermaak et al., 2011) 27 C1 - Internal use Figure 2: Chemical structures of important phytosterols (Vermaak et al., 2011) Figure 3: Chemical structures of tocopherols (Constantinou, Papas and Constantinou, 2008) 28 C1 - Internal use 2.2 Profile and characteristics of the seed oils 2.2.1 Moringa (Moringa oleifera) The Moringa oleifera (MO) tree, also known as the Drumstick or Malunggay tree is one of thirteen Moringa species. It is native to the Himalayas in India but is also found and utilised in most parts of Asia and Africa (Sengupta and Gupta, 1970; Lalas and Tsaknis, 2002; Dhakad et al., 2019). This tree is found in dry tropics making it resilient to drought. MO trees produce fragrant white flowers and bear fruits in the form of long green pods that contain multiple seeds. These seeds are globular with leaf-like wings (Leone et al., 2016). The MO tree has traditionally been used for cooking oil, food, cosmetics, and medication. All parts of this plant, namely the leaves, fruits, flowers seeds and roots, are edible. This makes for a good source of crude fibre, minerals, protein, and nutrients (Verma and Nigam, 2014; Dhakad et al., 2019). The chemical profile and quality of the seed oil are comparable to olive oil and may be used as a cheaper alternative (Lalas and Tsaknis, 2002; Zhao and Zhang, 2013; Nadeem and Imran, 2016). MO oil, also known as Ben oil, has a fatty acid composition that includes a high level of MUFAs and a lower level of PUFAs (Mani, Jaya and Vadivambal, 2007). The MUFAs include mainly oleic acid, which has good oxidative stability that allows for longer storage, high-temperature frying, use in medicine and water treatment (Anwar and Bhanger, 2003). Saturated palmitic and stearic acid are present as well as the PUFAs linolenic and linoleic acids (Lalas and Tsaknis, 2002). MO oil has a high vitamin E content making the oil resistant to oxidative degradation once refined (Koheil et al., 2011). Other vitamins present include vitamin A and B. The seed oil has high pharmaceutical value as it is anti-inflammatory, antithrombotic, antihypertensive, antioxidizing, hepatoprotective, antibacterial, antifungal, antiseptic, antiepileptic and vasodilating (Cicero and Gaddi, 2001; Janaki, 2015; Nadeem and Imran, 2016; Ghafar et al., 2017). MO oil can also be used in treatment to lower 29 C1 - Internal use cholesterol levels, treat venomous bites, rheumatism, gout, skin damage such as cuts and burns as well as skin conditions such as eczema and psoriasis (Koheil et al., 2011; Ojiako and Okeke, 2013; Ghafar et al., 2017). MO oil has high cosmetic value as it is a non-drying and moisturising oil that is easily absorbed by the skin. The oil can thus be used as an active ingredient, a carrier, emollient, or preservative. It is used in a wide range of cosmetic formulations such as cleansers, massage oils, soaps, moisturisers, anti-aging products, lip balm, perfumes, scrubs, hot oil hair treatments and anti-hair fall lotions (Mehta et al., 2011; Warra, 2011; Nadeem and Imran, 2016). Figure 4 below is an illustration of the MO plants components and Table 1 contains a summary of the major physico-chemical characteristics literature values of MO seed oil. Figure 4: The pods, seeds and kernels of Moringa oleifera (Noh, 2018) 30 C1 - Internal use Table 1: Major Physico-chemical characteristics literature values of MO seed oil (Cold-pressed) Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Yellow liquid (Anwar and Bhanger, 2003; Ogbunugafo et al., 2011; Ruttarattanamongkol et al., 2014) Acid value (mg KOH/g) 0.19 ± 0.03 (Özcan et al., 2019) Saponification value (mg.KOH/g) 181.3 ± 3.67 (Özcan et al., 2019) Average molecular weight (g/mol) 928.30 (Özcan et al., 2019) Ester value (mg KOH/g) 181.11 ± 3.67 Calculated from AV and SV results (Özcan et al., 2019) Iodine value (g/100g) 67.53 ± 1.37 (Özcan et al., 2019) Peroxide value (mequiv/kg) 0.18 ± 0.07 (Özcan et al., 2019) Density (g/ml) (25 °C) 0.819 ± 0.013 (Özcan et al., 2019) Specific gravity (25 °C) nd Refractive index (25 °C) 1.4359 ± 0.0001 (Özcan et al., 2019) Highest content fatty acid Oleic acid (Özcan et al., 2019) nd, no data 2.2.2 Marula (Sclerocarya birrea) Marula trees are deciduous trees native to many parts of sub-Saharan Africa such as Ethiopia, Ghana, Nigeria, Zimbabwe, South Africa, Angola, Zambia, Botswana, Namibia, Mozambique etc. (Ngorima, 2006; Borochov- Neori et al., 2008; Russo et al., 2013; Hetta, 2016). They are commonly found in woodland habitats in sandy loam soil (Borochov-Neori et al., 2008; Vermaak et al., 2011; Francis and Tahir, 2016) Marula trees are leafless during winter, produce pink flowers during spring and bear edible fruit during summer (Hetta, 2016). The small Marula fruits are oblong shaped with a thick yellow skin and sticky flesh rich in vitamin C. Normally 2-3 soft edible seed kernels are encapsulated in each fruit (Ogbobe, 1992; Vermaak et al., 2011). Oil extraction is challenging due to the shape of the hard brown seed within the nut (Cheikhyoussef, 2018). Marula has commonly been used for food (nutrition), alcoholic beverage, cosmetics, leather treatment, meat preservation and medicine (Mariod, Matthaus and 31 C1 - Internal use Eichner, 2004; Athar and Nasir, 2005; Kleiman, Ashley and Brown, 2008). The roots, leaves and bark have traditionally been used to treat conditions such as dysentery, fevers, malaria, diarrhoea, rheumatism, sore eyes, infertility, headaches, toothache, body pains and are used in infant's nose drops (Zimba et al., 2005). Extracts have antifungal, antioxidant, antibacterial, astringent, anticonvulsant, antiatherogenic, antihyperglycemic, anti-inflammatory, and aphrodisiac properties (Zimba et al. 2005; Francis and Tahir, 2016). Marula oils phytosterol content consists mainly of β-sitosterol and smaller amounts of 5-avenasterol. These phytosterols add antioxidative properties to the oil and also indicate that this seed oil is a potential source of protein (Mariod, Matthaus and Eichner, 2004). It contains unsaturated arachidonic acid which is important in the human diet (Mariod, Matthaus and Eichner, 2004). The seed oil contains mainly oleic, palmitic and stearic fatty acids (Vermaak et al., 2011). It however has a low content of tocopherols making it a poor source of vitamin E. Despite a deficiency in the antioxidant vitamin E, Marula oil is a rich source of oleic acid making it very stable due to the MUFAs antioxidative properties (Mariod, Matthaus and Eichner, 2004). Marula oil is medium-rich and silky to touch with low odour, it penetrates well making it an excellent moisturiser (Zimba et al., 2005). This non-irritating oil is ideal for use in cosmetic products as it improves skin hydration, reduces skin redness and relieves skin irritation (Zimba et al. 2005; Vermaak et al., 2011; Hetta, 2016). Cosmetic applications of this oil include massage oils, face oil, pomades, soaps, after-sun lotions, aftershaves and makeup removers (Zimba et al. 2005; Francis and Tahir, 2016; Cheikhyoussef, 2018). Figure 5 below is an illustration of the Marula fruit and Table 2 contains a summary of the major physico-chemical characteristics literature values of Marula seed oil. 32 C1 - Internal use Figure 5: Sclerocarya birrea (Marula) (Hetta, 2016) Table 2: Major Physico-chemical characteristics literature values of Marula seed oil (Cold-pressed) Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Light yellow liquid (Cheikhyoussef, 2018) Acid value (mg KOH/g) 5.16 ± 0.16 (Cheikhyoussef, 2018) Saponification value (mg.KOH/g) 187.94 ± 0.48 (Cheikhyoussef, 2018) Average molecular weight (g/mol) 895.52 ± 2.28 (Cheikhyoussef, 2018) Ester value (mg KOH/g) 182.77 ± 0.63 (Cheikhyoussef, 2018) Iodine value (g/100g) 85.28 ± 29.30 (Cheikhyoussef, 2018) (Robinson, Lukman and Bello, 2012) Peroxide value (mequiv/kg) 1.16 ± 2.52 (Cheikhyoussef, 2018) (Robinson, Lukman and Bello, 2012) Density (g/ml) (25 °C) nd Specific gravity (25 °C) 0.93 ± 0.96 (Cheikhyoussef, 2018) (Robinson, Lukman and Bello, 2012) Refractive index (25 °C) 1.46 ± 0.003 (Cheikhyoussef, 2018) (Robinson, Lukman and Bello, 2012) Highest content fatty acid Oleic acid (Mariod, Matthaus and Eichner, 2004; Nwabuebo, 2017; Cheikhyoussef, 2018) nd, no data 33 C1 - Internal use 2.2.3 Kalahari melon (Citrullus lanatus) The Kalahari melon is a trailing herb native to Namibia and Botswana. This plant has broad leaves and produces yellow flowers. It bears yellow or green fleshy fruits that vary in size and encapsulate black or pale-yellow seeds. Kalahari melon is commonly found on river banks, dry lakes, or disturbed areas (Vermaak et al., 2011). This plant is commonly used for cooking oil, food (nutrition), water source, cosmetics and medication (Nyam et al., 2010; Lendelvo et al. 2012; Cheikhyoussef, 2018). Tar extracted from the seeds are used for the treatment of scabies and skin tanning. Kalahari melon is also used to treat conditions such as herpes lesions, urinary diseases, fever, acne, venereal sores, leg ulcers and for prevention of cancer and coronary heart disease (Cho, 2004; Mariod et al., 2009; Vermaak et al., 2011). Kalahari melon seed oil is known to have a high content of tocopherols (Nyam et al., 2010). This high vitamin E level gives the oil natural antioxidative properties which make the seed oil very stable and thus ideal for industrial, cosmetic, nutritional, and pharmaceutical purposes (Vermaak et al., 2011). The seed oil contains essential fatty acids, the most abundant being linoleic acid followed by oleic, palmitic and stearic acids. Amongst the phytosterols present in the oil, β-sitosterol is the most abundant followed by campesterol and stigmasterol, which adds to the oil's natural antioxidant capacity (Vermaak et al., 2011; Hetta, 2016). This seed oil is typically used in cosmetic formulations as an emollient due to its light texture. The high essential fatty acid content makes this oil very moisturising and nourishing to the skin, therefore, restoring the skin's elasticity (Vermaak et al., 2011). Due to its properties, Kalahari melon seed oil is used in cosmetic formulations such as after-sun skincare, moisturizers, hair lotions, massage oils and soaps (Nyam et al., 2010; Vermaak et al., 2011; Cheikhyoussef, 2018). Figure 6 below is an illustration of Kalahari melon fruit and Table 3 contains a summary of the major physico-chemical characteristics literature values of Kalahari melon seed oil. 34 C1 - Internal use Figure 6: Citrullus lanatus (Kalahari melon) (Hetta, 2016) Table 3: Major physico-chemical characteristics literature values of Kalahari melon seed oil (Cold-pressed) nd, no data Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Yellow liquid (Cheikhyoussef, 2018) Acid value (mg KOH/g) 5.16 ±0.16 (Cheikhyoussef, 2018) Saponification value (mg.KOH/g) 189.26 ±1.40 (Cheikhyoussef, 2018) Average molecular weight (g/mol) 889.30 ±6.58 (Cheikhyoussef, 2018) Ester value (mg KOH/g) 188.16 ± 1.41 (Cheikhyoussef, 2018) Iodine value (g/100g) 117.64 ± 1.96 (Cheikhyoussef, 2018) Peroxide value (mequiv/kg) 2.12 ± 0.052 (Cheikhyoussef, 2018) Density (g/ml) (25 °C) nd Specific gravity (25 °C) 0.922 ± 0.001 (Cheikhyoussef, 2018) Refractive index (25 °C) 1.4726 ± 0.001 (Cheikhyoussef, 2018) Highest content fatty acid Linoleic acid (Cheikhyoussef, 2018) 35 C1 - Internal use 2.2.4 Mongongo/ Manketti (Schinziophyton rautanenii) The Mongongo/ Manketti tree is a large deciduous and dioecious tree. This tree is native to Southern Africa, namely Angola, Namibia, Botswana, South Africa and Zambia, and is found in wooded hill and deep sand habitats (Atabani et al., 2014). It has dark-green leaves and small pale- yellow flowers appearing in spring (Vermaak et al., 2011). This tree bears small egg-shaped green fruits covered in fine hairs, these fruits fall from the tree in autumn and ripen on the ground (Vermaak et al., 2011). The ripe fruit has a sweet soft flesh and relatively large off-white seed. This tree is traditionally a source of food, cosmetics and medicine (Vermaak et al., 2011). The fatty acid profile of the seed oil resembles that of maize oil. It consists mainly of the PUFAs linoleic, linolenic and arachidonic acid, followed by oleic, palmitic, stearic and erucic acids (Vermaak et al., 2011). The seed oil is rich in phytosterols and natural proteins including γ- tocopherol and 𝛽-sitosterol, making it nutritious and protective to the hair and skin (Athar and Nasir, 2005; Mitei et al., 2009; Hetta, 2016). The high vitamin E content provides the oil with high oxidative stability and long shelf life, making it useful to the pharmaceutical, food and cosmetic industry (Zimba et al. 2005; Juliani et al., 2007; Nyam et al., 2010). The seed oil may be used for the treatment of eczema, atopic disorders, reduction of itching, redness, wrinkles, age spots, scarring, burns and the prevention of keloids (Zimba et al., 2005; Cheikhyoussef, 2018). This is due to the oil being anti-inflammatory, antibacterial, rich in antioxidants and promoting skin renewal (Zimba et al., 2005; Vermaak et al., 2011). Cosmetic formulations using this light texture oil that easily absorbs into the skin include hair lotions, cleansers, aromatherapy products, moisturisers and emollients (Graz, 2002; Juliani et al., 2007; Vermaak et al., 2011; Kivevele and Huan, 2014). Figure 7 below is an illustration of Mongongo fruit and Table 4 contains a summary of the major physico- chemical characteristics literature values of Mongongo seed oil. 36 C1 - Internal use Figure 7: Schinziophyton rautaneii (Mongongo) (Hetta, 2016) Table 4: Major physico-chemical characteristics literature values of Mongongo seed oil (Cold-pressed) Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Light yellow (Juliani et al., 2007; Cheikhyoussef, 2018) Acid value (mg KOH/g) 1.83 ± 0.33 (Juliani et al., 2007; Cheikhyoussef, 2018) Saponification value (mg.KOH/g) 187.66 ± 1.52 (Cheikhyoussef, 2018) Average molecular weight (g/mol) 896.9 ± 7.30 (Cheikhyoussef, 2018) Ester value (mg KOH/g) 185.61 ± 1.55 (Cheikhyoussef, 2018) Iodine value (g/100g) 130.15 ± 1.63 (Juliani et al., 2007; Cheikhyoussef, 2018) Peroxide value (mequiv/kg) 5.60 ± 4.12 (Juliani et al., 2007; Cheikhyoussef, 2018) Density (g/ml) (25 °C) nd Specific gravity (25 °C) 0.923 ± 0.003 (Cheikhyoussef, 2018) Refractive index (25 °C) 1.48 ± 0.00 (Juliani et al., 2007; Cheikhyoussef, 2018) Highest content fatty acid Linoleic acid (Cheikhyoussef, 2018) nd, no data 2.2.5 Baobab (Adansonia digitate L.) The Baobab tree, native to Madagascar, is commonly found in parts of sub-Saharan Africa such as Malawi, Zimbabwe, Mozambique, South Africa, Benin, Senegal, Cameroon, Kenya, Uganda, Tanzania etc. (Vermaak et al., 2011). This slow-growing deciduous tree is the largest 37 C1 - Internal use succulent in the world found in hot arid regions with low rainfall. Leaves are hand shaped (digitate) and are only present for three months in a year whereas the white hanging flowers, consisting of five petals, are present for only two months of the year (Vermaak et al., 2011; Komane et al., 2017). The seeds are found in the velvety hair covered egg-shaped fruits (Gebauer, et al. 2006). The baobab tree has multiple purposes as each part of the tree; namely the fruit pulp, seeds, leaves, flowers, roots, and bark; have a valuable use. Local communities use this versatile tree for food, medicine, cosmetic purposes as well as for raw materials for other purposes such as fertilizer and clothing. The fruit pulp is especially nutritious as it is high in vitamin C. Baobab is used to treat numerous conditions such as fever, dandruff, diarrhoea, coughs, haemoptysis, dysentery, worms, kidney diseases, burns and other skin conditions as well as scurvy related symptoms (Watt and Breyer-Brandwijk, 1962; Zimba et al., 2005; Vermaak et al., 2011; Mahomoodally and Ramjuttun, 2017). These anti-inflammatory, antimicrobial, and anti-viral properties make baobab important to the pharmaceutical industry (Chindo et al., 2010; El- Nagerabi et al., 2013; Komane et al., 2017). This conditioning oil contains many vitamins including vitamins A, D3, E and F (Nkafamiya et al., 2007). Vitamins A and F(polyunsaturated) are responsible for the rejuvenation and renewal of cell membranes, therefore, repairing the skin. Vitamin E is an antioxidant, making the oil very stable and giving the seed oil antiaging activity. The oil contains palmitic, linoleic and oleic essential fatty acids (Andrianaivo-Rafehivola, A.A. Blond et al., 1993; Mahomoodally and Ramjuttun, 2017). These components make baobab oil an excellent moisturizer that can also reduce eczema and psoriasiss (Wren and Stucki, 2003; Athar and Nasir, 2005; Zimba et al., 2005). The major sterols present are β-sitosterol, stigmasterol, and campesterol, which adds to the anti-oxidative properties of the seed oil (Vermaak et al., 2011). The rich seed oil is non-irritating and non-sensitizing which can be used for normal and dry skin (Wren and Stucki, 2003). This oil is highly penetrating and 38 C1 - Internal use can soften dry skin, improve its elasticity and tone without a greasy residue (Wren and Stucki, 2003; Zimba et al., 2005; Hetta, 2016). Baobab seed oil is therefore used in bath oil preparations, moisturisers, emollients, massage oils, hot oil soaks and many other cosmetic applications (Wren and Stucki, 2003; Zimba et al., 2005; Vermaak et al., 2011) This fatty acid rich oil is therefore very important to the cosmetic industry (Chindo et al., 2010; Komane et al., 2017). Figure 8 below is an illustration of Baobab fruit and Table 5 contains a summary of the major physico-chemical characteristics literature values of Baobab seed oil. Figure 8: Adansonia digitatal (Baobab)(Hetta, 2016) Table 5: Major physico-chemical characteristics literature values of Baobab seed oil Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Yellow liquid (Cissé et al., 2018) Acid value (mg KOH/g) 18.827 ± 0.309 (Cissé et al., 2018) Saponification value (mg.KOH/g) 233.587 ± 0.478 (Cissé et al., 2018) 39 C1 - Internal use Average molecular weight (g/mol) 720.50 (Cissé et al., 2018) Ester value (mg KOH/g) 214.76 ± 0.36 (Cissé et al., 2018) Iodine value (g/100g) 99.113 ± 0.528 (Cissé et al., 2018) Peroxide value (mequiv/kg) 2.091 ± 0.579 (Cissé et al., 2018) Density (g/ml) (25 °C) 0.911 ± 0.04 (Cissé et al., 2018) Specific gravity (25 °C) 0.943 (Chindo et al., 2010) Solvent extraction Refractive index (25 °C) 1.464 ± 2.8 (Cissé et al., 2018) Highest content fatty acid Oleic acid (Muthai et al., 2019)(Zimba et al., 2005) 2.2.6 Hemp (Cannabis sativa L.) Hemp is a part of the Cannabis sativa species which falls under the Cannabaceae family. Hemp is a dioecious, flowering herb with digitate leaves and two lower leaflets. The small flower buds contain and protect many seeds each encapsulated in a shell (Citti et al., 2018). The Hemp plant is very versatile and can be used in food, medicine, paint, cosmetics, fibres and textiles, animal feed etc. The seeds have a relatively high oil content which is very rich in PUFAs mainly linoleic, linolenic, and arachidonic acid. The oil has a favourable ratio of 3:1 omega 3 to omega 6 PUFA, which is optimal for nutrition, however making it chemically unstable (Athar and Nasir, 2005; Smeriglio et al., 2016; Citti et al., 2018; Tan et al., 2018). The far lower MUFA content includes oleic acid and saturated stearic acid. The seed oil contains moderate to high amounts of vitamin E and phytosterols making the oil a source of natural antioxidants (Oomah et al., 2002; Mikulcová et al., 2017; Citti et al., 2018). Hemp seeds contain no cannabinoids, therefore, any trace amounts of cannabinoids present in hemp seed oil are impurities caused by the extraction process, which is usually cold-pressing (Vogl et al., 2004; Citti et al., 2018). Hemp seed oil is used in the treatment of skin conditions such as neurodermatitis and psoriasis (Vogl et al., 2004). The oil's fatty acid profile makes the oil anti-inflammatory, antibacterial, accelerate healing of https://en.wikipedia.org/wiki/Dioecious https://en.wikipedia.org/wiki/Flowering_plant https://en.wikipedia.org/wiki/Herb https://en.wikipedia.org/wiki/Leaflet_(botany) 40 C1 - Internal use skin wounds and regenerate skin and hair (Tan et al., 2018). Hemp seed oil is ideal for cosmetic applications due to its rich PUFA content giving it emollient and moisturizing properties (Vogl et al., 2004). Hemp seed oil is used in cosmetic formulations aimed at skin protection via lipid barrier restoration as well as hair moisturizing and growth products such as shampoos and hair treatments (Athar and Nasir, 2005; Tan et al., 2018; Paul et al., 2019). Figure 9 below is an illustration of the Hemp plants components and Table 6 contains a summary of the major physico- chemical characteristics literature values of Hemp seed oil. Figure 9: Hemp seeds (left), a young hemp plant (centre), and a mature flowering hemp plant (Smith, 2018) 41 C1 - Internal use Table 6: Major physico-chemical characteristics literature values of Hemp seed oil (Cold-pressed) Physico-chemical characteristic Average literature value Literature sources Colour and state (25 °C) Yellow-green liquid (Al Jourdi et al., 2019) Acid value (mg KOH/g) 1.7 ± 0.6 (Mikulcová et al., 2017) Saponification value (mg.KOH/g) 197.6 ± 4.5 (Mikulcová et al., 2017) Average molecular weight (g/mol) 851.72 (Mikulcová et al., 2017) Ester value (mg KOH/g) 195.9 ± 4.46 (Mikulcová et al., 2017) Iodine value (g/100g) 155.8 ± 1.9 (Mikulcová et al., 2017) Peroxide value (mequiv/kg) 0.90 (Al Jourdi et al., 2019) Density (g/ml) (25 °C) 0.908 (Al Jourdi et al., 2019) Specific gravity (25 °C) nd Refractive index (25 °C) 1.474 (Al Jourdi et al., 2019) Highest content fatty acid Linolenic acid (Al Jourdi et al., 2019) nd, no data 2.3 Extraction techniques 2.3.1 Cold-press extraction Cold-press extraction of seed oil involves the application of immense pressure on pre-treated seeds to separate the liquid and solid phase (Zuorro et al., 2014). The cold press machine has one inlet where seeds are fed into, two outlets that release the oil product and a protein-rich by- product known as a seed press cake (Singh and Bargale, 2000). Moringa seed press cakes may be used as a fertiliser (Emmanuel et al., 2011), animal feed and as a natural coagulant in water purification (Ndabigengesere and Narasiah, 1998; Mehta et al., 2011; Oladeji et al., 2017). Process parameters include pressure, feeding rate, restriction dye diameter and rotation speed of propeller (Çakaloğlu, Özyurt and Ötleş, 2018; Chemat et al., 2019). This technique is referred to as cold-press extraction as no additional heat is applied during the process. Care is however taken for some oils to limit the amount of heat generated by friction during the process to prevent oxidation reactions and loss of volatile compounds, such as phenolics (Parry et al., 2005; Nwabuebo, 2017; Çakaloğlu, Özyurt and Ötleş, 2018). Cold-pressing has the 42 C1 - Internal use advantage of being fast, cheap, low-energy and safer as products obtained are free of harmful solvents making it more environmentally friendly (Rotkiewicz, Konopka and Zylik, 1999; Balami, 2007; Ethiopia, 2018; Özcan et al., 2019). The oil produced is high in quality with better nutritional properties than refined oils and very little to no refinement is needed except for washing with water, filtering or centrifugation (Agus Bin et al., 2014; Çakaloğlu, Özyurt and Ötleş, 2018). Cold-pressing, however, has the disadvantages of lower productivity, consistency, yields and selectivity compared to other extraction methods (Çakaloğlu, Özyurt and Ötleş, 2018; Ethiopia, 2018). Figure 10 below illustrates Cold-pressing oil extraction. Figure 10: Modified illustration of cold-pressing (Vaughn et al., 2018) 2.3.2 Soxhlet extraction Soxhlet extraction is one of the traditional extraction techniques to which other extraction techniques are compared (Wang and Weller, 2006). Experimental parameters for this technique include solvent choice (like dissolves like), the contact area of the analyte with solvent (particle size), amount of solvent (solute to solvent ratio), temperature (diffusion rate), the drying time of sample (moisture content), and extraction time (Daroch, 43 C1 - Internal use Geng and Wang, 2013; Balcıoğlu, 2015; Nwabuebo, 2017; Çakaloğlu, Özyurt and Ötleş, 2018) In this study, hexane will be used as the extraction solvent. Hexane is commonly used for extracting vegetable oils for its non-polar nature, efficiency and reliability compared to other solvents such as benzene, toluene and chloroform (Reverchon and De Marco, 2006; Nwabuebo, 2017). The procedure involves extraction by repeated washing (percolation) with an organic solvent, in this case, hexane. The prepared seed sample is placed in filter paper in an extraction chamber situated between a round bottom flask containing hexane, and a condenser. The solvent is heated such that it volatilises and moves into the condenser where it condenses and drips into the extraction chamber. The excess solvent surrounding the sample overflows and drips back down into the solvent flask. During each cycle of the extraction process, a portion of the analyte dissolves in the solvent. After many cycles, the analyte is concentrated in the solvent flask and can then be separated from the solvent using a rotary evaporator (Ethiopia, 2018). Soxhlet extraction has numerous advantages such as high yields, high efficiency, low costs, recyclable solvent, simple equipment and no need for filtration of the oil (Pradhan et al., 2010; Palafox et al., 2012; Çakaloğlu, Özyurt and Ötleş, 2018). The disadvantages, however, are that the solvents are ecologically harmful, toxic and flammable (Bhattacharjee, Singhal and Tiwari, 2007). Recovery and recycling of solvent are difficult and therefore use high amounts of hence residual solvent in the final product is common (Palafox et al., 2012; Nwabuebo, 2017; Çakaloğlu, Özyurt and Ötleş, 2018; Tan et al., 2018). Figure 11 below illustrates the Soxhlet extraction apparatus. 44 C1 - Internal use Figure 11: Illustration of Soxhlet extraction apparatus (Bhutada et al., 2016) 2.3.3 Ultrasound-assisted extraction Ultrasound-assisted extraction is a relatively modern extraction technique that makes use of ultrasound waves to assist the extraction process. Ultrasound waves are above the human hearing range with frequencies over 20 kHz. Propagation of these high-frequency waves above the local tensile strength of the liquid cause a negative pressure to develop which induce vapour bubbles that result in macroturbulence and shear forces as they collapse. Mechanical effects such as cavitation bubbles, mixing, vibration, and pulverization, therefore, take place. Ultimately, this mechanical disruption aids extraction as the cell walls of the sample is agitated and damaged which allows easier permeability of the extraction solvent and therefore intensified mass transfer. Cavitation also results in a chemical disruption by the formation of free radicals that trigger chemical reactions to take place that further aid the extraction process. Ultrasound- assisted extraction is therefore a greener extraction process as the 45 C1 - Internal use mechanism allows for reduced extraction time and lower temperatures ideal for sensitive compounds. Factors that affect extraction efficiency include frequency, solvent type, solvent volume sample size and extraction time (Thirugnanasambandham, 2018; Mwaurah et al., 2020). The ultrasound instrument is either in the form of an ultrasound bath or an ultrasound probe depending on the application as illustrated in Figure 12. Table 7 below contains average literature values for MO oil extraction yields and efficiency. Figure 12: Schematic illustration of an ultrasonic bath and ultrasonic probe(Behzadnia, Moosavi-Nasab and Tiwari, 2019) Table 7: Average literature values for MO oil extraction yields and efficiency Method of extraction % Oil yield Literature values Literature sources % Efficiency Literature values Literature sources Soxhlet extraction (hexane) 38.3 (Lalas and Tsaknis, 2002) 100.00 ± 1.00 (Nwabuebo, 2017) 35.30 ± 1.14 (Nwabuebo, 2017) 35.3 (Tsaknis et al., 1998) 46 C1 - Internal use 35.7 ± 2.4 (Tsaknis et al., 1999) 29.12 (Dinesha et al., 2018) 40.12 (Zhao and Zhang, 2013) 40.39 ± 1.15 (Anwar and Bhanger, 2003) 41.47 (Ogbunugafo et al., 2011) 39.16 ± 0.19 (Zhong et al., 2018) 30.8 ± 2.19 (Abdulkarim et al., 2005) Average: 32.87 ±10.11 Cold-press extraction 14.43 (Nwabuebo, 2017) 60.91 (Nwabuebo, 2017) 25.1 (Lalas and Tsaknis, 2002) 65.54 Estimate based on (Lalas and Tsaknis, 2002) 26.3 (Tsaknis et al., 1998) 74.50 Estimate based on (Tsaknis et al., 1998) 25.8 ± 2.6 (Tsaknis et al., 1999) 72.27 Estimate based on (Tsaknis et al., 1999) Average: 22.1 ± 5.67 Average: 68.34 ± 6.23 Ultrasound- assisted extraction 38.1 (Buddin et al., 2018) 59 (Thirugnanasamban dham, 2018) 53.8 ± 0.43 (Mohammadpo ur et al., 2019) M. peregrina oil 35.77 ± 0.04 (Zhong et al., 2018) Average: 42.56 ± 9.81 Shaker method extraction 27.19 ± 0.91 (Nwabuebo, 2017) 77.01 ± 0.55 (Nwabuebo, 2017) 47 C1 - Internal use 2.4 Characterization and analytical techniques 2.4.1 Gas Chromatography-Mass Spectrometry Gas Chromatography-Mass Spectrometry (GC-MS) can be utilised as both a quantitative and qualitative technique. This combination technique can separate mixtures of volatile and semi-volatile compounds and selectively detect them. The GC instrument separates chemical mixtures into individual compounds using heat. The volatilised compounds are carried through a column with an inert gas, usually helium. As the separated compounds exit from the column opening, they flow into the MS detector. Individual compounds can then be identified by their unique mass using an MS library of known compound spectra. Quantitation is made possible by using calibration curves of known standards. GC-MS can be used for applications such as drug and pesticide detection and quantification (Sneddon, Masuram and Richert, 2007). The solvent selected to dissolve the samples and rinse the column depends on the polarity of analytes present. In some cases, a mixture of solvents is required for complex matrices. In this study and those similar, GC-MS is used to identify and/or quantify tocopherol, sterols and/or fatty acids. Since these are not volatile compounds, a derivatisation process is done to samples before analysis. For fatty acid analysis in particular many researchers use reference standards to identify and quantify the fatty acids present in a sample. From the total fatty acid concentrations or relative percentages, percentages of saturated fats (SAFA), Trans-fats (TFA), Monounsaturated fats (MUFA), polyunsaturated fats (PUFA), and total unsaturated fats (TUFA) can be determined. This type of data can be used as characterisation references or fingerprint data to which future data can be compared (Danish and Nizami, 2019). 48 C1 - Internal use Cheikhyoussef (Cheikhyoussef, 2018) for example used GC-MS methods described by Du and Ahn (Du and Ahn, 2002) to analyse major tocopherol, sterol and fatty acid content in various Namibian seed oils, namely manketti, marula, Ximenia, !nara and melon seed oil. These compounds' identification and/or quantification are of great importance to many industries. Cheikhyoussef in particular was profiling these seed oils for the food industry and the effect extraction techniques had on quality. Highest total tocopherol content found was 205.64 mg/100g (Manketti; Soxhlet extracted), 29.72 mg/100g (Marula; traditionally extracted), 10.75 mg/100g (Ximenia; traditionally extracted), 46.10 mg/100g (!Nara, Soxhlet extracted) and 74.39 mg/100g (Melon; cold-pressed). The highest stigmasterol (53.11 mg/100g) was found in traditionally extracted Marula nut oil and the highest β- sitosterol content (682.43 mg/100g) was found in Soxhlet extracted Manketti nut oil. Major fatty acids found were linoleic (31.2- 32.2%) acid, α-eleostearic (24.2-35.7%) acid (Manketti), oleic (66.6- 67.6%) acid (Marula), oleic (46.3-44.1%) acid, ximenynic (6.5-12.0%) acid (Ximenia), linoleic (52.6-57.0%) acid, oleic (10.5-17.7%) acid (Melon) and linoleic (53.1-54.5%) acid, oleic (12.8-13.9%) acid (!Nara) (Cheikhyoussef, 2018). Figure 13 bellow illustrates the main components of the GC–MS instrument. Figure 13: Schematic plot of the main components of the GC–MS instrument (Fullan, 2015) 49 C1 - Internal use 2.4.1 Nuclear Magnetic Resonance Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique that can be used qualitatively or quantitatively. It provides information on atomic environments based on the distinct resonance frequencies displayed by nuclei, in this case, hydrogen and carbon, in a strong magnetic field. The identity, purity and molecular structure of samples can be determined using this technique. NMR is a sensitive non-destructive technique that requires minimal sample preparation. NMR spectroscopic results are in the form of NMR spectra, chemical shift (𝛿) vs intensity (Sherazi and Mahesar, 2015). NMR is a simple technique that can be used for the fast screening of large numbers of samples and the development of a database of authentic products to detect adulteration (Popescu et al., 2015). In a study by Popescu and co-workers, 1H and 13C NMR analysis was used for multiple pure and vegetable oil blends to determine the saturated fatty acids, oleic acid, linoleic acid, linolenic acid and iodine value. The major aim being to determine the capabilities of NMR and chemometrics to be used in the quality assessment of vegetable oils in terms of their botanical origin. What was found is that the largest contribution to the total organic signal obtained for an oil sample was given by the 1.30 ppm signal (53%) representing the methylene groups of all the fatty acids, followed by 2.03 ppm, 0.87 ppm and 5.37 ppm signals with contributions of approximately 10%, the lowest signal being given by the 1.02 ppm group (terminal methyl group of linolenic acid) with percentages around 0.68%. The integral positions and intensities give information on the oils' overall saturation and allowed Popescu and co-workers to correctly group the oils with the same level of saturation which correlates to their iodine values. Similarly, Mitei and co-workers used NMR to determine the relative compositions of the saturated, monounsaturated and diunsaturated fatty acids and their average chain lengths. This was done using the relative http://chem.ch.huji.ac.il/nmr/qc.htm#impurities http://chem.ch.huji.ac.il/nmr/identify.htm http://chem.ch.huji.ac.il/nmr/identify.htm https://www.sciencedirect.com/topics/chemistry/nmr-spectrum 50 C1 - Internal use sizes of the NMR signals integrals for the signals corresponding to the allylic, diallylic and methyl protons using Holmback’s equations. Other techniques including GC-MS were used in conjunction to confirm fatty acid content. It was found that NMR fairly accurately elucidates the fatty acid content in oils except for some poor resolution of the oleoyl and linoleoyl signals in the 13C-NMR spectrum (Mitei et al., 2008). Figure 14 below illustrates the components of the NMR instrument. Figure 14: Schematic diagram of an NMR spectrometer(Rankin et al., 2014) 2.4.2 Ultraviolet-visible spectroscopy Ultraviolet-visible (UV-Vis) spectrometers are one of the most important analytical instruments. UV-Vis spectrometry is known for its simplicity, versatility, speed, accuracy and cost-effectiveness. Spectroscopy is related to matters interaction with light which involves the radiation being absorbed, transmitted, scattered, reflected or it can excite fluorescence. When matter absorbs radiation, there is an excitation of electrons present in the solution producing a distinct spectrum. The principle behind UV-Vis 51 C1 - Internal use follows the Beer-Lambert Law which states that when monochromatic radiation passes through a homogenous solution in a cell, the decreasing rate of the radiation intensity along with the thickness of the absorbing solution is proportional to the concentration of the solution and the incident radiation. This law is expressed through the equation: A = log (I0/I) = ECL, where I0 is the intensity of the incident radiation, I is the intensity of transmitted radiation, C is the concentration of the solute, L is the length of the sample cell and E is the molar absorptivity. UV-Vis spectroscopy is mainly used for the quantitative determination of different compounds in a solution. It may also be used for applications such as determining the purity of a substance and identifying unknown compounds by comparing its spectrum to the spectrum of a reference compound(ThermoSpectronic, 2012). Nwabuebo (Nwabuebo, 2017) as well as Delfan-Hosseini and co-workers used UV-VIS to study the radical scavenging activity, of various oils extracted in different ways, towards 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. This was done to elucidate their antioxidant properties and therefore their oxidative stability. This simple colourimetric analysis involved monitoring the decrease in absorbance at 517 nm for the prepared oil samples in comparison to a reference sample. In the study conducted by Nwabuebo, % Inhibition, which correlates directly to antioxidant content, was as follows for both marula and moringa seed oil: Aqueous extraction at 37 °C > Aqueous extraction at 60 °C> Screw press> Hexane extraction (shaker)> Hexane extraction (Soxhlet) Hexane (Nwabuebo, 2017). In the study conducted by Delfan-Hosseini and co- workers antioxidant content in Purslane seed oil was found to be in the following order: microwave pre-treatment cold press extracted> Solvent extracted> Cold press extracted. In both cases, it was concluded that the oil extraction method used does indeed affect the oils' antioxidant properties and therefore oxidative stability. According to Nwabuebo, this is 52 C1 - Internal use due to differences in solvent and temperature between the different extraction methods that affect natural antioxidant content present thus leading to a lower scavenging ability/ % inhibition (Nwabuebo, 2017). Figure 15 below illustrates the components of a UV-Vis instrument. Figure 15: Schematic diagram of a UV-Vis spectrometer(Sobarwiki, 2013) 2.4.3 Differential scanning calorimetry Differential scanning calorimetry (DSC) is a thermoanalytical technique that has been used in different ways over the years to determine and monitor several different characteristics and trends in oils and fats (Tan and Che Man, 2000). It is a fast and sensitive technique that requires very little sample for analysis (Differential Scanning Calorimetry for Routine Analysis, 2015). DSC works on the basic principle that both a sample and reference sample are taken through a temperature programme and maintained at the same temperature throughout. It is the energy needed by the system to maintain both sample and reference at the same temperature that allows for the identification and quantification of physical 53 C1 - Internal use transformations that are taking place in the sample by measuring the enthalpies associated with transitions and reactions and the temperatures at which these take place. Differences in heat flow take place when the analyte/sample absorbs or releases heat due to thermal effects such as melting, chemical reactions, crystallization, vaporization, polymorphic transitions, and many other processes (Differential Scanning Calorimetry for Routine Analysis, 2015). Due to the complex chemical profiles of oils, they do not have specific melting and freezing/ crystalizing temperature but rather thermal profiles showing associated changes in enthalpy and therefore phase transitions (Tan and Che Man, 2000). By determining the oxidation onset temperature of oils, the thermal stability can be measured and old or used oils can be distinguished from new or fresh oils (Differential Scanning Calorimetry for Routine Analysis, 2015). Mohammadpour and co-workers made use of DSC to study the difference in thermal behaviour between Moringa peregrina extracted in different ways, namely ultrasound-assisted extraction and Soxhlet extraction. Melting profiles of the two samples both produced two exothermic peaks with onset temperatures of −12.5 °C and 6.4 C for UAE as well as −21 °C and 6.2°C for the Soxhlet method. Cooling profiles showed a single endothermic peak for both samples with onset temperatures of -37.4 °C for UAE and -35.9 °C for the Soxhlet extracted oil. This indicated that the UAE sample contained a higher degree of saturated fatty acids. In conclusion, they found that the different extraction techniques had no significant effect on the thermal properties of M. peregrina oil (Mohammadpour et al., 2019). Nyam and co-workers however used the DSC to compare thermal properties amongst different oils namely bittermelon, Kalahari melon, kenaf, pumpkin and roselle seed oils. Melting and heating profiles indicated that bittermelon seed oil crystallized at a lower onset temperature of -12.10 °C, and melted at a higher onset temperature of 54 C1 - Internal use 37.52 °C, in comparison to the other seed oils. A significant difference in melting and cooling profiles could be seen between different oils allowing this technique to be used as an identification analysis for unknown seed oil samples. According to the general trend that samples with a high content of saturated fatty acids produce DSC melting and cooling profiles with higher temperatures as compared to oil samples with a high content of unsaturated fatty acids, bittermelon seed oil contains a higher content of saturated fatty acids compared to the other oils studied (Nyam et al., 2009). Figure 15 below illustrates the components of a DSC instrument. Figure 16: Schematic diagram of DSC(Bibi et al., 2015) 2.4.4 Fourier transform- Infrared spectroscopy Fourier transform infrared spectroscopy (FT-IR) is a simple and fast analytical technique that can give accurate and reliable data. Fragments of structure can be elucidated due to characteristic vibrations, and quantitative information can be determined using peak intensity as a direct relationship to concentration. Characteristic absorption peaks at specific 55 C1 - Internal use wavelength ranges take place due to the unique vibrational or rotational energy transformations a chemical bond in a molecule undergoes from the ground to the excited state when exposed to infrared light. The spectrum produced is thus a series function of absorbed energy responses. Due to these characteristic infrared absorption frequencies, each molecule, except for isomers, has a unique fingerprint spectrum. This can be used to identify molecules and confirm the purity of a sample (Laboratories, 2015; Li et al., 2019). Oji and Vivian (Oji and Vivian, 2020) used FT-IR to determine the compounds present in coconut, avocado and carrot oil. From the three FT- IR spectra it was evident that all three oils possessed similar major compounds. These functional groups include the O-H group of carboxylic acids aromatics and C-O stretch in esters. The aromatic overtone of ring bends band peak at 1744.4cm-1 was a deep peak for coconut and carrot oil however a narrow peak for avocado oil. Due to the striking similarities in the FT-IR spectra of coconut and carrot oil, it was very clear that coconut oil was used as the base oil in the extraction of carrot carrier oil (Oji and Vivian, 2020).Oladipo and Betiku (Oladipo and Betiku, 2019) used FT-IR as one of the characterisation techniques to assess the difference between Moringa oleifera seed oil extracted by Soxhlet extraction using three different solvents namely ethyl acetate, ethanol and n-hexane. The results showed equivalent peaks in all the spectra, indicating that the choice of solvent did not affect the functional groups of the oil samples (Oladipo and Betiku, 2019). Figure 17 below illustrates the components of an FT-IR instrument. 56 C1 - Internal use Figure 17: Schematic diagram of FT-IR(Bibi et al., 2015) CHAPTER 3: AIMS AND OBJECTIVES 3.1 Aims This study aimed to investigate the chemical composition of commercial Moringa, Marula, Kalahari melon, Mongongo, Baobab, and Hemp seed oil for use in cosmetics. This was achieved through the following objectives: 3.2 Objectives • Extraction of Moringa seed oil using Cold-press, Soxhlet, Ultrasound-assisted and Shaker extraction techniques. • Determination of the chemical profile of commercial Moringa, Marula, Kalahari melon, Mongongo, Baobab and Hemp seed oils. • Understand from the chemical and physical composition, the strength of each oil in cosmetic industry applications. • Trial formulation of a cosmetic product(s) incorporating these commercial seed oils using modified existing formulation. 57 C1 - Internal use 3.3 Motivation The significance of this research study stems from the fact that high variability exists amongst seed oil literature currently available. An in-depth study on the chosen oils using the same seed source for extraction and the same analysis techniques significantly reduced variability and therefore allowed one to make more accurate comparisons between parameters of the different oils (Tan and Che Man, 2000). This broad study looked at MO oil extracted by four different methods, as well as profiling these oils alongside underutilised African oils in comparison to well-known commercial vegetable oils such as olive and sunflower oil. This study aimed to increase the database of knowledge available on underutilised African oils to assess their economic importance. Increasing commercialisation of these oils will benefit rural communities involved in harvesting, encourage maintenance of the natural environment, provide more export products and alternative natural ingredients for the cosmetic industry which will overall benefit our bioeconomy (Zimba et al., 2005; Juliani et al., 2007; Vermaak et al., 2011). These non-traditional seed oil sources, if managed correctly, can satisfy the ever-growing demand for this important renewable commodity by supplementing the world's current vegetable oil supplies (Mitei et al., 2008; Janaki, 2015). CHAPTER 4: MATERIALS AND METHODS 4.1 Materials and reagents All reagents and solvents used in this study were purchased from Merck as an analytical grade. Hexane, anhydrous sodium sulphate, ethanol, phenolphthalein, sodium hydroxide, potassium hydroxide, hydrochloric acid, calcium oxide, chloroform, iodine, potassium iodide, sodium thiosulphate, starch, acetic acid, 2, 2-diphenyl-1-picrylhydrazyl, ascorbic acid, ethanol, 5- 𝛼 cholestane, campesterol, 𝛽- sitosterol, 58 C1 - Internal use stigmasterol, 𝛼, 𝛽, 𝛾 𝑎𝑛𝑑 𝛿 tocopherol standards, Supelco 37 components FAMES mix. Moringa oleifera (MO) seeds and remaining seed oils were locally sourced. Cosmetic bases were purchased from Nautica organics. Working solutions were prepared by appropriate dilution of the stock solutions with solvent. 4.2 Extraction of Moringa oleifera seed oil 4.2.1 Sample preparation MO seeds were sourced from Lefakong Farming based in Hammanskraal. The seeds were manually cracked and deshelled to obtain the kernel. The kernels were then air-dried over two days to reduce water content. The kernels (~1 kg) were then ground into a fine powder using a domestic blender and stored at -20 °C until extraction. Figure 18 below shows the state of the MO seed during sample preparation. Figure 18: Illustration of the MO sample preparation steps from A- Whole seed, to B- De-shelled kernel and lastly C-Kernel/seed powder 4.2.2 Cold-press extraction Cold-press extraction of MO oil was carried out at the Innovation Hub by Lefakong Farming. Whole MO seeds were used for extraction, after which the oil was filtered to remove impurities and stored in an amber glass bottle. In Figure 19 below the MO oil, the cold-pressing process is explained. A- Power was switched on; pressure and heat settings were adjusted. B- Whole MO seeds were added to a feed hopper. C- Milling/ 59 C1 - Internal use screw component then fed the seeds into the main component of the machine in a uniform manner. D- A controlled amount of seeds then entered the squeezing/ pressing chamber. E- The MO oil was then pressed out from the whole seeds. F- Crude oil drips out from below squeezing/ pressing chamber into a container fitted with a sieve to catch large seed material. G- Leftover seed material form solid seed cakes that were expelled at the end of the pressing process. H- Seed cake by- product to be used for animal feed. From this point, oil would then be filtered and packaged. A B 60 C1 - Internal use G F E D C H 61 C1 - Internal use Figure 19: Illustrations of the MO oil Cold-pressing process (A-H) 4.2.3 Soxhlet extraction About 45 g of seed kernel powder was placed in filter paper inside a Soxhlet extractor with cot