ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA (AMADUMBE) A TRADITIONAL CROP FOOD IN KWAZULU-NATAL Ronalda McEwan 056257 Thesis Submitted to the Department of Biochemistry and Microbiology, Faculty of Science University of ZuluJand in Partial FuliIllment of the Requirements for the Degree Philosophy Doctor (ph.D) in Biochemistry at the University of Zululand. Promoter: Prof AR Opoku Co-Promoters: Prof T Djarova Dr OA Oyedeji KwaDlangezwa 2008 DEDICATION I can do all things through Christ which strengthened me Philippians 4:13 ABSTRACT Colocasia esculenta 1. Schott belongs to the family Aracea and is grown for its edible corms as a staple food throughout subtropical and tropical regions of the world Amadumbe (the Zulu name for Colocasia esculenta) is consumed by and holds an important place in the diet oflocal rural people in Kwazulu-Natal, South Africa. Three Amadumbe phenotypes were evaluated for their nutritional qualities. Like all known tubers, the locally grown Amadume contained high carbohydrate levels, adequate protein and low lipid content. Essential fatty acids (linoleic and linolenic) were identified as components of the Amadumbe lipids. Amadumbe was generally low in mineral content, apart from potassium and magnesium levels that were relatively high. Some anti-nutrients (protease inhibitors, lectin, phenolic compounds, alkaloids, oxalates, phytates, cyanogens and saponin) present in Amadumbe were also identified and quantified The anti-nutrient levels were generally low and thus may not pose an immediate effect on the health of consumers. Reduction of the anti nutrients through processing (cooking, frying, roasting) was observed to enhance the nutritional value of these tubers. However, their presence suggests that a steady consumption may lead to toxic levels. Two proteins (AI and B2) with a-amylase inhibitor activity, and a steroidal saponin (gamma-sitosterol) were extracted and partially characterised. The a-amylase inhibitors were extracted and partially purified through ammonium sulphate precipitation and chromatographic fractionation on diethylaminoethyl (DEAE)-Sephacel and Sephadex G-lOO. The molecular weights of the two inhibitors were estimated to be 17 000 and 19 000 dalton, respectively. The inhibitors were fairly heat-stable, with optimum activity at 40? C, pH 6.0. Both inhibitors showed activity against mammalian a amylases, but were devoid of activity against fungal amylases. Inhibitor A also showed activity against plant amylases. The steroidal saponin extracted from Amadumbe was characterized through TLC, HPLC, GC-MS, IR and NMR spectroscopic analysis and identified to be gamma- 1 sitosterol, an isomer of beta-sitosterol which is known to have a variety of high biological activity. Studies of the effect of beta-sitosterol on absorptive and digestive enzymes in Sprague-Dawley rats revealed that oral administration of beta-sitosterol had no apparent gross or microscopic lesions in the liver, kidney or small intestine. The administered ~-sitosterol significantly decreased serum aspartate aminotransferase (ALT) and alanine aminotransferase (AST) levels. Na+/K+-AlPase and intestinal disaccharidases activities were also significantly reduced in beta-sitosterol fed rats. These results do suggest that even though Amadumbe is a neglected crop in South Africa, it is a highly nutritional crop; the consumption of it could be beneficial to diabetic and hypertensive patients. 11 ACKNOWLEDGEMENTS The work presented in this thesis has been carried out at the University of Zululand, KwaDlangezwa, Empangeni, South Africa I would like to thank all the people that contributed to this thesis. ? My promoter, Professor Andy Opoku (Department of Biochemistry), for your support and encouragement. Thank you for keeping me focused during the stressful times. ? My co-promoters Professor T Djarova (Department of Biochemistry) and Dr OA Oyedeji (Department ofChemistry). ? Professor Djarova, thank you for directing and providing me with invaluable advice. ? Bola if it wasn't for you helping me with characterization of saponin I would never have finished - thank you for assisting and encouraging me when my moral was low. ? My darling husband Andrew for all your help, especially with feeding the rats! Also your love and endless support, always being positive and putting me and the completion of this thesis first. ? My daughter, Chante for being so patient with me while I worked such long hours - thank you, you are a little star. ? My mom and dad for all the love and support. ? All the technical help: ? Anita, Brett and Dilip at the University of Kwazulu-Natal for providing your apparatus and expertise and help. ? Piet Oosthuizen for providing Amadumbe from Makatini Research farm. ? Lina Wallis from Capital Lab for providing me with all the chemicals. 1ll ? My colleagues Kayode, Stranger, Deepo and Nejo - thank you for all the fun we had while struggling through all the experiments. ? Ralph, Sphiwe, Forgiveness, Thobi and Nomthi for your contributions to complete this thesis. ? Sasha Pay for all the assistance and help with the statistics. ? Linda Nienaber for editing this document for me - you were an angel. Ronalda McEwan IV TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS LIST OF ABBREVIATIONS LIST OF FIGURES LIST OF TABLES CHAPTERl GENERAL INTRODUCTION I SECTION A PROXIMATE ANALYSIS AND SCREENING FOR SOME ANTI-NUTRIENTS IN PROCESSED AND UNPROCESSED COLOCASIA ESCULENTA SUMMARY 3 CHAPTERA-l: LITERATURE REVIEW A 1.1 INTRODUCTION 4 A 1.2 COLOCASIA ESCULENTA (AMADUMBE) 5 A1.3 FATlY ACIDS 10 AI.4 ANTI-NUTRIENTS 11 A. 1.4. I Proteinase Inhibitors (PI) - Trypsin Inhibitor 11 A. 1.4.2 Amylase Inhibitors 15 A. 1.4.3 Lectins 16 A.1.4.4 Totalpolyphenols 17 A. 1.4.5 A1kaloids 20 A. 1.4.6 Oxalate 22 A. 1.4.7 Phytate 23 A 1.4.8 Cyanogens 26 A. 1.4.9 Saponins 29 A1.5 EFFECT OF PROCESSING ON NUTRIENTS AND ANTI-NUTRIENTS 30 AI.6 AIM AND OUTLINE OF SECTION A 32 v CHAPTER A-2 : MATERIALS AND METHODS A2.1 lNfRODUCTION 33 A2.2 MATERIALS A2.2.1 Food material 33 A2.2.2 Reagents 33 A2.2.3 Special equipment 35 A2.3 MElHODS A2.3.1 Sample preparation 35 A.2.3.2 Processing techniques 35 A.2.3.3 Proximate composition 36 A.2.3.4 Fatty acids 36 A2.3.5 Mineral analysis 38 A2.3.6 DETERMINATION OF ANTI-NU1RIENTS A2.3.6.1 Trypsin Inhibitor 38 A.2.3.6.2 Amylase Inhibitor 38 A.2.3.6.3 Lectin 39 A.2.3.6.4 Total polyphenols 40 A2.3.6.5 Tannins 40 A.2.3.6.6 Flavonoid 40 A.2.3.6.7 Cyanogens 40 A2.3.6.8 Phytate 41 A.2.3.6.9 Alkaloids 41 A.2.3.6.1O Oxalate 41 A2.3.6.911 Saponin 41 CHAPTERA-3: RESULTS A3.1 lNfRODUCTION A3.2 PROXIMATE COMPOSmON A3.3 MINERAL ANALYSIS A3.4 FATTY ACIDCOMPOSmON A3.5 ANTI-NUTRITIONAL FACTORS CHAPTER A-4 : DISCUSSION A4.1 lNfRODUCTION VI 43 43 47 49 59 66 A.4.2.1 A.4.2.2 A.4.3 A.4.4 PROXIMATE COMPOSmON MINERAL ANALYSIS FATTY ACID COMPOSmON ANTI-NUfRITIONAL FACTORS 66 69 70 71 CHAPTER 5: CONCLUSION A.5.1 NUfRITIONAL AND ANTI-NU1RITIONAL EVALUATION 78 A.5.2 PROCESSING AND ANTI-NU1RITIONAL FACTORS 79 REFERENCES 81 SECTION B PURIFICATION AND CHARACTERIZATION OF a-AMYLASE INBIBITORAND SAPONIN PRESENT IN COLOCASlA ESCULENTA SUMMARY 118 CHAPTER B1-1 : LITERATURE REVlEW (a-AMYLASE INBIBITOR) Bl-1.1 INTRODUCTION 119 Bl-1.2 a-AMYLASE INHIBITOR 119 Bl-1.3 AIM AND OUTLINE OF SECTION B-1 124 CHAPTER Bl-2 : MATERIALS AND METHODS Bl-2.1 INTRODUCTION 125 Bl-2.2 MATERIALS 125 Bl-2.3 METHODS B1-2.3.1 Extraction and purification ofa-amylase inhibitor 126 Bl-2.3.1.1 Ion-exchange chromatography 127 Bl-2.3.1.2 Gel Chromatography 127 Bl-2.3.2 EnzymefInhibitor assay 127 BI-2.3.3 Molecular weight determination 128 Bl-2.3.4 Kinetic studies 128 CHAPTERBl-3: RESULTS Bl-3.1 INTRODUCTION 129 BI-3.2.1 EXTRACTION AND PURIFICATION OF a-AMYLASE INHIBITOR 129 Bl-3.22 KINETIC STUDIES 134 vu CHAPTER 8-1-4 : DISCUSSION Bl-A.l INTRODUCTION BI-4.2 ISOLATION OF a-AMYLASE INHIBITOR Bl-4.3 KINETIC STUDIES Bl-4.3.1 Action ofinhibitors on different a-amy1ases Bl-4.3.2 Effect oftemperature on the inhibitor activity BI-4.3.3 Effect ofpH on the interaction ofamylase with inhibitor CHAPTER B2-1 : LITERATURE REVIEW (SAPONIN) B2-1.1 INTRODUCTION B2-1.2.1 SAPONIN-CHEMISlRY B2-1.2.2 Triterpenoid saponin B2-1.2.3 Steroidal saponin B2-1.2.4 Biological activity B2-2.1.3 AIM AND OUTLINE OF SECTION B-2 CHAPTER B2-2 : MATERIALS AND METHODS B2-2.1 INTRODUCTION B2-2.2 PLANT MATERIAL B2-2.3 METHODS B2-2.3.1 Saponin extraction and isolation B2-2.3.2 Chromatographic methods B2-2.3.3 Spectroscopy CHAPTER B2-3 : RESULTS B2-3.1 INTRODUCTION B2-3.2 EXTRACTION AND PURIFICATION OF SAPONINS CHAPTER B2-4 : DISCUSSION B2-4.1 INTRODUCTION B2-4.2 SAPONIN CHAPTER 8-5 : GENERAL CONCLUSION BSI a-AMYLASE INHIBITOR "VUl 137 137 137 137 138 139 140 140 142 144 145 146 147 147 148 149 150 152 152 158 158 163 SECTION C EFFECTS OF INGESTED BETA-SITOSTEROL ON DIGESTIVE AND ABSORPTIVE ENn'MES IN RATS SUMMARY CHAPTER C-l : LITERATURE REVIEW C.1.l INTRODUCTION C.1.2.1 Stereochemical structure C.l.2.2 Pharmacochemistry and Pharmacokinetics c.1.2.3 Biological activities c.1.3 DIGESTIVE ENZYMES C.1.3.1 Disaccbaridases CHAPTER C-2 : MATERIALS AND METHODS C.2.1 INTRODUCTION C.2.2 MATERIALS AND ME1HODS C.2.3 ETHICAL CONSIDERATION B.5.2 REFERENCES C.1.3.2 C.1.4 C.2.2.2 C.2.2.3 C.2.2.4 C.2.2.5 C.2.2.6 SAPONIN ATPase AIM AND OUTLINE OF SECTION C Solubilisation ofbeta-sitosterol PRELIMINARY TOXICITY TEST EXPERIMENTAL TEST Measurement ofenzyme activities STATISTICAL ANALYSIS 163 162 181 182 182 183 184 186 188 190 191 191 191 191 192 193 194 195 CHAPTER C-3 : RESULTS C.3.1 INTRODUCTION C.3.2 PRELIMINARY TOXICITY TEST 196 C.3.2.1 Food and water consumption 196 C.3.2.2 Body weight changes 197 C.3.2.3 Clinical signs 198 LX C.3.3 EXPERIMENTAL TEST C.3.3.1 Food and water consumption 198 C.3.3.2 Body weight changes 199 C.3.3.3 Clinical signs 201 C.3.3.4 Heamatology tests 202 C.3.3.5 Pathology and organ weights 203 C.3.3.6 Digestive enzyme activities in small intestine C.3.3.6.1 ATPase activity 208 C.3.3.6.2 Disaccharidase activity 205 CHAl'TER C-4 : DISCUSSION C4.1 . INTRODUCTION 212 C4.2 EXPERIMENTAL TEST 212 CHAPTERC-5: CONCLUSION 218 REFERENCES 219 CHAPTER 2: GENERAL CONCLUSION 2.1 INTRODUCTION 234 2.2 NUTRITIONAL AND ANTI-NU1RITONAL EVALUATION 234 2.3 CHARACTERIZATION AND NU1RITIONAL EVALUATION OF 235 SELECTED ANTI-NUTRIENTS IN AMADUMBE 2.4 SUGGESTIONS FOR FUTURE WORK 236 APPENDIX A: APPENDIXB APPENDIXC APPENDIXD APPENDIXE .PREPARATION OF REAGENTS DETAILS OF METHODOLOGY OLEIC ACID STANDARD CERTIFICATE FROM ETHICS COMMITTEE POSTERS PRESENTED AT CONFERENCES x 237 242 258 259 260 ACAT ALT AI AlA AOAC apoB AST ATP ATR BAPNA BP BPH BW CCK DM DNS El-MS EP ER EW FAO FP FW GA GC-MS HCN HPLC HPLC-UV IR KCN KZN LIST OF ABBREVIATIONS Acyl-coenzyme A cholesterol acyltransferase alanine aminotransferase amylase inhibitor amylase inhibitor activity Association ofOfficial Analytical Chemists apolipoprotein B aspartate aminotransferase adenosine triphosphate attenuated total reflectance benzoyl-DL-arginine-p-nitroaniline Esikhawini boiled purple benign prostatic hypertrophy Esikhawini boiled white cholecystokinin dry matter dinitrosaliccyclic acid electron impact-mass spectrometry Esikhawini purple endoplasmic reticulum Esikhawini white Food and Agriculture Organization Esikhawini fried purple Esikhawini fried white glycoalkaloids gas chromatography-mass spectrometry hydrogen cyanide high-performance liquid chromatography High-Pressure Liquid Chromatography with Ultra-Violet Detector infra-red potassium cyanide Kwazulu-Natal Xl MakP Makatini purple MakW Makatini white MNU methyInitrosourea MtP Mtubatuba purple MtW Mtubatuba white MUFA monounsaturated fatty acid ND no date NMR nuclear magnetic resonance PI proteinase inhibitor PVP polyvinylpolypyrrolidone RP Esikhawini roasted purple RW Esikhawini roasted white TAN tropical ataxic neuropathy TG triacylglycerol TIll T-helper-l TIA trypsin inhibitor activity TMS tetramethylsilane WHO World Health Organisation Xli 22 24 Figure A1-1 Figure Al-2: Figure Al-3 Figure Al-4 Figure Al-5 Figure Al-6 Figure Al-7: Figure Al-8 Figure Al-9 Figure A1-lO Figures A3-1 (a-d, and a1-d1) Figure A3-2 FigureA3-3 Figure A4-1 Figure Bl-2.1 Figure Bl-3.1 Figure Bl-3.2 Figure Bl-3.3 Figure Bl-3.4 Figure Bl-3.5 LIST OF FIGURES Diagram showing the differences in corm structure between these 5 botanical varieties. The aboveground portion ofColocasia esculenta (Amadumbe or 7 Taro) Amadumbe chips with mango atchar 9 Mode ofaction ofsoybean trypsin inhibitors on the pancreas 14 Structure ofa flavonoid (flavonol) 19 Structures of two major glycoalkaloi~ found in potatoes: a- 21 solanine and a-ehaconine Structure ofoxalic acid Possible interactions ofphytic acid with minerals, proteins and starch. Structures ofsome common cyanogenic glycosides. 27 Structure ofone ofthe saponins present in soybeans 29 Chromatographic separation of Amadumbe (Colocasia esculenta) 50 fatty acids Phytochemical screening for flavonoids in the unprocessed and 62 processed tubers Phytochemical screening for alkaloids in the unprocessed tubers 65 The covering percentage for each mineral in 1 g of an Amadumbl 70 tuber Extraction protocol for isolating a-amykase inhibitor from 126 Amadumbe tubers Ammonium sulphate precipitation of the Amadumbe crude 130 Ion-exchange chromatography ofextract on DEAE-Sephacel 131 Sephadex G-lOO column chromatography of fraction A and B 132 after ion-exchange chromatography on DEAE-Sephacel Standard Log Molecular Weight graph using gel filtration 133 Effect of temperature on the activity of Colocasia esculenta a- 135 amylase inhibitors Xlll Figure BI-3.6 Figure B2-1.1 Figure B2-1.2 Figure B2-1.3 Figure B2-1.4 Figure B2-2.1 Figure B2-3.1 Figure B2-3.2 Figure B2-3.3 Figure B2-3.4 Figure B2-3.5 Figure B2-4.1 Figure B2-4.2 Figure B2-4.3 Figure B2-4.4 Figure CI-1 Figure Cl-2 Figure C3-1 Figure C3-2 Figure C3-3 Figure C3-4 Figure C3-5 Figure C3-6 Figure C3-7 Figure C3-8 Effect of pH on the stability ofAmadumbe a-amylase inhibitor 136 Types ofsaponin 141 Pathway to biosynthesis oftriterpenes 142 Basic aglycone (sapogenin) skeletons -triterpene 143 Basic aglycone (sapogenin) skeletons - steroidal 145 Extraction protocol for obtaining native sapomns from 148 Amadumbe tubers Thin-layer chromatographic examination of saponin compounds 153 from an Amadumbe (Colocasia esculenta) tuber Infrared spectrum of saponin B1 fraction of white Colocasia 154 esculenta Chromatogram of saponin B1 from Amadumbe anaylsed by GC- 155 MS Chromatographic profile ofsaponin B1 analysed by GC-MS 156 Major fragmentation pattern of sitosterol irrespective of the 157 isomeric form Numbering ofsterol carbons 159 The structural formula ofgamma-sitosterol 159 The structural formula ofbeta-sitosterol 160 The structural formula ofgamma- and beta-sitosterol 161 Hydrolysis of ATP to ADP, the fundamental mode of energy 188 exchange in biological systems The sodium-potassium exchanger, which establishes the ionic 189 concentration balance that maintains the cell potential. Growth rates ofrats fed diets containing beta-sitosterol 197 Body weight gains of male Sprague-Dawley rats 199 Body weight/food consumed ofmale Sprague-Dawley rats 200 Male Sprague-Dawley rats used for experimental tests 201 Representative photomicrographs of histology ofliver 205 Representative photomicrographs of histology of kidney 206 Representative photomicrographs of histology of duodenum and 207 ileum Lower and upper intestinal Na+/K+-ATPase activity in rats 208 XlV Figures C3-9 Figure B-1 The activity of small intestine disaccharidases ofthe rat groups. 209 Burette packaging for starch percolation 243 xv TableA3-la TableA3-lb TableA3-lc TableA3-2a TableA3-2b TableA3-3a TableA3-3b TableA3-3c Table A-3-4a TableA3-5 TableA3-4b TableA3-4c Table BI-3.1 TableBI-3.2 Table C2-1 Table C3-l Table C3-2 LIST OF TABLES The moisture and ash content (g/IOOg DM) of processed and unprocessed 44 Amadumbe (Colocasia esculenta) tubers The crude fat and crude protein composition (g/ lOO per cent DM) in 45 unprocessed and processed Amadumbe Carbohydrate content of unprocessed and processed Colocasia esculenta 46 tubers Macronutrient profile of unprocessed and processed Colocasia esculenta 47 (mg/lOOg DM) Micro-element contents of unprocessed and processed Colocasia 48 esculenta (mg/lOOg DM) Yields ofcrude fat ofAmadumbe extracted with 49 methanol-ehloroform mixture (2: I, v/v) Iodine value of Amadumbe lipid extract 49 Fatty acids identified in Amadumbe Colocasia esculenta 58 Trypsin inhibitor activity (mg of trypsin inhibited/g DM) and a-amylase 59 inhibitor activity ofunprocessed and processed Amadumbe tubers Inhibitory effect ofcrude extract on other proteases 60 The phenolic compounds in the processed and unprocessed Amadumbe 61 tubers The levels of some anti-nutritional factors in processed and unprocessed 63 tubers (Colocasia esculenta) from Zululand Purification ofamylase inhibitors from Amadumbe 129 Effect ofa-amylase inhibitors present in Amadumbe against 134 amylases from different sources Composition ofadministered materials for preliminary toxicity test groups 192 Average food and water consumption in rats over a six-day period in 196 preliminary studies Average food and water consumption of rats during a 14-day period of 198 experimental studies XVI Table C3-3 TableC3-4 Summary of serum biochemistry and haematological parameters ofmale 202 Sprague-Dawley rats orally administered with beta-sitosterol for 14 days Effect of beta-sitosterol on absolute and relative organ weights of male 203 Sprague-Dawley rats xvu CHAPTER 1: GENERAL INTRODUCTION Nutritional value is the main concern when a plant is considered as food source. However, endogenous toxic :fuctors characteristic of plant material can also affect the content of nutrients. These toxic factors act as anti-nutrients and adversely affect the organism. Anti nutrients are chemicals which have been evolved by plants for their own defence, among other biological functions. Anti-nutrients reduce the maximum utilization of nutrients (especially proteins, vitamins and minerals), thus preventing optimal exploitation of the nutrients present in a food and decreasing the nutritive value (Ugwu and Oranye, 2006). Anti-nutrients vary in chemical structures, ranging from amino acids to proteins; from simple amines to a1kaloids, glycosides and many phenolic compounds. The biological effects ofall these chemicals are diverse and complex. When man ingests plant foods to meet nutritional needs, a wide variety of these non nutrient phytochemicals are ingested at the same time. Processing is expected to inactivate these anti-nutritional factors and increase the availability of bioactive compounds. However, the health risk to consumers of large quantities of residual anti-nutrients cannot be ruled out In sub-Saharan Africa, approximately three quarters of the population live in rural areas. Most of their dietary energy comes from staple cereals, such as maize. Given the limited resources and restricted access to different foods in rural societies, most African communities have developed diets that maximize the use of local foodstuffs. The advancements in grain production have not brought significant benefits to areas where tuber crops are the major staples. Therefore, emphasis should be placed on such tuber crops as Amadumbe (Colocasia esculenta), which is a staple food in many developing nations of West-Africa (as taro/cocoyam), Asia and the Pacific (as cocoyam). Amadumbe (Colocasia esculenta) is widely grown in the sub-tropical parts of South Africa as a traditional food I crop. Amadumbe is not extensively commercialized at present, but is mostly grown in rural areas or on small fimns in Kwazulu-Natal, South Africa. Literature abounds with research carried out on Colocasia esculenta species in other parts of the world, but little or no information is available regarding the composition, structural activity and biochemical mechanisms of anti-nutritional factors in local Colocasia esculenta (Amadumbe). Studies investigating these factors could help the government in formulating food and nutrition policies for South Africa. The overall hypothesis of this study was, therefore, that if the constituents of various nutritional and anti-nutritional factors inherent in Colocasia esculenta (Amadumbe), a traditional crop food grown in Kwazulu-Natal, were identified, this tuber could be more efficiently utilized in food and nutrition policies for South Africa. The project was divided into three specific aims to meet the overall objective: ? to determine the proximate composition, the mineral and the anti-nutrient content of Amadumbe from Kwazulu-Natal, South Africa, in order to compare the factors with those observed in Colocasia esculenta from other areas of the world. At the same time, the best processing method for maximum elimination of the anti-nutritional factors would be identified; ? to isolate and characterize some of the screened anti-nutritional factors to determine the structural activity and biochemical properties of these toxicants occurring naturally in Amadumbe tubers; ? to perform a nutritional evaluation using rats with a specific, identified anti-nutritional factor from Colocasia esculenta to determine the biological effects of the anti nutritional factoL 2 SECTION A PROXIMATE COMPOSITION AND SOME ANTI NUTRIENTS IN PROCESSED AND UNPROCESSED COLOCASIA ESCULENTA SUMMARY Amadumbe (Colocasia esculenta) is widely grown in the subtropical parts of South Africa as a subsistence crop. Like most root crops, Amadumbe is high in carbohydrate content with low levels of protein and lipids. Amadumbe contain some essential fatty acids. Preliminary screening of Amadmnbe revealed the presence of some anti-nutrients (substances in food of plant origin that interfere directly with the absorption of nutrients). Processing (boiling, frying and roasting) of Amadumbe showed a reduction in the content of these anti-nutritional factors (amylase inhibitor, trypsin inhibitor, oxalate, alkaloids, saponin, phytate and total phenols). Of the three different treatments, boiling appeared to be the most effective in reducing levels of all the investigated anti-nutrients in the white variety of Amadmnbe. Any of the domestic processes used in this research work could be employed detoxifying most of the investigated anti-nutritional factors in Colocasia esculenta. 3 CHAPTER A-I LITERATURE REVIEW ~l.l Introduction A community will accept certain foods as suitable for their consumption and these foods, because of custom and the people's partiality towards them, become regarded as traditional food crops. There are many examples of plant foods consumed as traditional dietary staples that are indigenous to Africa - for example: cassava, yam, plantain, sweet potato, millets and sorghum (Food and Agriculture Organization of the United Nations, 1997). Amadumbe (Colocasia esculenta) is a non-indigenous specie widely grown in the sub tropical parts of South Africa as a subsistence crop. Locally developed cultivars are used as a dietary source of starch. As a tuber crop, Amadumbe makes a significant contribution to the diet of local people in Kwazulu-Natal, where it is easily available. Several authors have evaluated the chemical composition of whole corms and cormels of taro, also known as cocoyam [Colocasia esculenta var. Schott of the tropics and subtropics in Africa (Sefa-Dedeh and Agyir-Sackey, 2004; Oscarsson and Savage, 2007)], but there is little or no information available on the nutritional quality of Amadumbe (Colocasia esculenta var Schott), the variety that is traditionally used in Kwazulu-Natal, South Africa. Anti-nutrients are compounds that limit the digestion and absorption of nutrients and result in reduced bioavailability of nutrients and flatulence production (Brune et al. 1989, Medoua et al., 2007). Technological processes (for example, cooking) partially eliminate most anti-nutrients so that their acute toxicity is, as a rule, mild (polomy, 1997; Adeparusi, 2001; West et al., 2007). However, the possible health risk to those consuming food with high quantities of residual anti-nutrients cannot be ruled out. This 4 section of the study therefore investigates the nutritional and anti-nutritional status of processed and unprocessed Amadumbe. A.I.2 CohJcasiJl esculenta (Amadwnbe) Amadumbe (Zulu name) belongs to the genus Colocasia, within the subfamily Colocasioideae of the monocotyledonous family Araceae. Taro or cocoyam is the common name for edIble aroids which are important staple foods in many parts of this world (Chay-Prove and Goebel, 2004). Onwueme (1999) noted the problems arising from the taxonomy of the genus Colocasia owing to its complicated vegetative propagation. Purseglove (1972, cited in Onweume, 1999) identified two major botanical varieties: i) Colocasia esculenta (L.) Schott var. esculenta; ii) Colocasia esculenta (L.) Schott var. antiquorum (Schott) Hubbard & Rehder, which IS synonymous with C. esculenta var. globulifera Engl. & Krause. (a) Colocasia esculenta var. esculenta (b) Colocasia esculenta var. antiquorum Figure AI-I: Diagram showing the differences in corm structnre between these two botanical varieties (a) Colocasia esculenta var. esculenta (a) and (b) Colocasia esculenta var. Antiquorum (Hawaiian kalo, 2007) 5 Plants of the genus Colocasia are edIble aroids with large leaves and one or more food storing underground stems (corms). The corm is made up of starchy ground parenchyma with a thick brown skin consisting of central circular leaf scares and scales (Lee, 1999). Onwueme (1999) observed that the Colocasia esculenta Yar. esculenta is made up of a central corm, bulky and cylindrical in shape, with not many cormels (Fignre AI-la), whereas Colocasia esculenta var. antiquorum has a number of large cormels arising from a small, central, globe-shaped corm (Fignre Al-Ib). Amadumbe is classified as Colocasia esculenta var. Schott (Van Wyk and Gericke, 2000). Taro (Amadnmbe) has been descnbed as a palatable, glabrate, annual herb. The leave blades (laminae) are large, 250 to 850 mm in length, 200 to 600 mm in width and 275 to 300 mm in thickness. The leaf laminae are carried on long, erect leafstalks (Fignre AI-2). The leaf shape is described as being complete and lanceolate, with the apex tapering to a concave point (Lee, 1999). Colocasia esculenta is best adapted to a warm, moist environment, ideally tropical or subtropical areas with long frost free periods. Amadumbe requires an average daily temperature above 21? C for normal production. High humidity with well-distributed summer rainfall or supplemental irrigation is ideal, partly because of the large, transpiring surfaces of Amadumbe laminae. Crops are normally grown in low-lying areas ranging from sea level to 1200 m elevation, only where moist conditions and stable temperatures generally prevail (Onwueme, 1999). Amadumbe is highly tolerant of saturated soil conditions, such as those found in wetlands, and artificial drainage of these soils is seldom required. Amadumbe cnltivation does little harm to the wetland if it is restricted only to the less sensitive parts of the wetland. This presumes that artificial fertilizers and pesticides are not used and tillage is carned out by hand. Excessive drainage should be avoided and the cnltivated area shonld be minimised. (Cnltivation, food & health: amadumbe, n.d.). 6 Figure AI-2: The aboveground portion of ColocasUl esculenta (Amadumbe or Taro) (Missouri Botanical Garden, n.d.) 7 Colocasta esculenta (L.) Schott is an ancient crop grown throughout Africa for its edible corms and leaves, as well as for its traditional uses. Matthews (2004, cited in Oscarsson and Savage, 2007) surmised that Colocasta esculenta (L.) Schott had its origins in the tropics between India and Indonesia and the Food and Agricultural Organization [FAO] (1992, cited in Oscarsson and Savage, 2007) commented that this plant had been grown in the South Pacific for hundreds ofyears. According to Bradbury and Nixon (1998), many Colocasia esculenta cultivars have a sharp, pungent taste and should not be eaten raw, because they can cause swelling of lips, mouth and throat. To enable comfortable ingestion, the tubers have to be thickly peeled and cooked over a long period (Saikai 1979; Crabtree and Baldry, 1982). Onayemi and Nwigwe (1987) identified the following constituents in Colocasia esculenta, once the causticity had been removed: digestIole starch, high-quality protein, essential amino acids, vitamin C, thiamin, riboflavin and niacin. In the rural areas ofKwazulu-Natal (SA), Amadumbe is the main starch source in cooked meals. Considered traditional African fare, the tubers are used in the same way as potatoes: fried as chips (Figure Al-3), mashed or barbequed whole (African affair, 2005). The young Amadumbe (cocoyam or taro) leaves can also be used as a vegetable (Aregheore and Perera, 2003): they are boiled for 15 minutes like spinach and are utilized as a supplement to maize. Leaves may be used in making salad and the Indian delicacy puripatha, often created in Durban, SA, is wrapped in Amadumbe leaves (Cultivation, food and health: arnadumbe, n.d). Lee (1999) observed that taro (Arnadumbe or cocoyam) is one of the only major staple foods where both the leaf and underground parts are used and have equal importance for human consumption. The excellent digestibility (98.8%) of the small starch grains of taro suggests efficient release of nutrients during digestion and absorption of this food (Lee, 1999). Much work has been done on Colocasta esculenta (L.) var. Schott. As mentioned before, cocoyam and taro are the other common names used for these edible aroids. Cocoyarns are grown for local consumption in West Africa and their corms and cormels are used in the same manner as yarns or potatoes for local dishes (Onayemi and Nwigwe, 1987). 8 Taro is produced in the Pacific Islands and parts of Asia as a main staple food crop. In New Zealand, it is developed as a secondary food crop. These plants contain digestIble starch, protein of good quality, vitamin C, thiamin, riboflavin, niacin and a high oxalate content (Onayemi and Nwigwe, 1987; Sefa-Dedeh and Agyir-Sackey, 2004; Perez et al., 2007; Catherwood et aI., 2007; Huang et aI., 2007; Oscarsson and Savage, 2007). Colocasia esculenta has been broadly investigated for proximate composition and anti nutrient screening, but the data were not comparable because of variations in genotypes, locations and experimental analysis. Plants are generally sensitive to environmental stress such as light intensity, rainfall, length of growing season, temperature and length of day, as well as agronomic factors such as soil fertility, weeds and plant density (McLean et al., 1974; Robertson et al., 1962; Singh et al., 1972). Information is scanty or non existent in literature on the presence of nutrients and anti-nutrients in Amadumbe. There are also no published studies on the effect of domestic cooking on the levels of anti nutrients in Amadumbe tubers of Zululand, South Africa. Figure AI-3 Amadumbe chips with mango atchar (www.woolworths.co.za) Traditional crops are developed in rural areas because they have a high nutritional value, they are resistant to drought and cultivation is easy as long as standard agricultural practices are followed. By utiling indigenous knowledge about Amadumbe, not only has the community gained economic benefits directly, but indigenous African fare has also 9 A.l.3 A.1.3.1 A.1.3.2 acquired an increased status. The upmarket South African chain store Woolworths has used indigenous knowledge provided to sell organically grown Amadumhe (Makgoba, 2004). Fatty acids Definition and structure Fatty acids (caIboxylic acids) often have a long, branchless tail, with carbon atoms linked in open chains (aliphatic). This chain can either be saturated or unsatmated. If the fatty acid does not contain any double bonds or other functional groups along the chain, it is termed saturated, whereas if it contains caIbon-eaIbon double bonds, it is called unsaturated. If only one double bond is present, it is mono-unsaturated and with two or more double bonds, it is polyunsaturated. Linoleic acid and alpha-linoleic acid are essential fatty acids - that is, those which are needed by the body. These acids cannot be synthesised by humans, but are widely distnbuted in plant oils (Fatty acid, 2008). Occurrence and food sources Fatty acids can occur as either free or bound forms. In the bound form, they are attached to other molecules, such as in triglycerides or phospholipids (Fatty acid, 2008). Although fatty acids are found in plants, animals and bacteria, they tend to be more complex in plants and bacteria than in most animals (Fatty acids, 2008). Essential fatty acids provide practically all the polyunsaturated fat needed by man. Omega-3 and Omega-6 fatty acids are drawn from a diversity of sources, which include fish, leafy vegetables, seeds from plants such as pumpkins, chia and sunflowers, a variety of vegetable oils (for example: fIaxseedl1inseed hemp, soya, canola/rapeseed) and nuts (for example: peanuts, walnuts) (Essential fatty acid, 2008). 10 A.1.4 Anti-nutrients Foods are complex substances that contain many chemical compounds, more than 50 of which are required to nourish the body. These nutrients include water, proteins, lipids, carbohydrates, minerals and vitamins. Additionally, most plant foods also consist of natural compounds or anti-nutrients that appear to function generally in defense against herbivores and pathogens. Anti-nutrients are potentially harmful and give rise to a genuine concern for human health in that they prevent digestion and absorption of vitamins, minerals and other nutrients. They may not be toxic as such, but can reduce the nutritional value of a plant by causing a deficiency in an essential nutrient or preventing thorough digestion when consumed by humans or animals (Pratlnba et aI., 1995). Several anti-nutritional factors are present in root and tuber crops and are partially neutralized during ordinary cooking. (Bhandari and Kawabata, 2004). The remaining anti-nutrients can, however, be responsIble for the development of serious gastric distress and may interfere with digestion of nutrients, which inevitably results in chronic deficits in absorption of nutrients (Kelsay, 1985; Jood et al., 1986; Brune et al., 1989). Anti nutritional factors include cyanogens, glycosides, saponins, phytate, enzyme inhibitors (trypsin and amylase inhIbitors), lectins (haemagglutinins), oxalate and total polyphenols. A.1.4.1 Proteinase inhibitors (PI) - Trypsin inhibitor Numerous biochemical and physiological processes involve proteolytic enzymes. Kowalska et at. (2007) list the following activities for which these enzymes are responsible: ? "cellular protein digestion; ? intracellular protein turnover associated with defense mechanisms; ? elimination of misfolded proteins; ? activation ofproenzyme, regulatory proteins and receptors; ? the release ofhormones and biologically active peptide; ? assembling processes; ? cellular differentiation and ageing; 11 ? seed development and mobilization of storage protein during seed gennination or seedling growth; ? pathogen suppression; and ? pest proteinases." Proteinase inhibitors which occur naturally play a crucial role in balancing body functions (Kowalska, 2(07). Protease can be inactivated by being blocked by inhibiturs andIor by proteins being broken down into simpler substances through hydrolysis (Troncoso et al., 2007). Proteinase inhibitors are proteins that bind to proteases, inhibiting proteolytic activity, and have been detected in animals, plants and microorganisms (Bhattacharyya et aI., 2007; Rawlings et al., 2004). Zhang et al. (2004) observed the proteinase inhibitors accwnulate at high levels in many plants becanse of attacks by bacteria and fungi, wounds and plant hormones. Bhattacharyya et al., (2007) identified 59 individual families of proteinase inhibitors, which could be classified mainly as serine, cysteine, aspartic or metallo inhibitors. By the 1930s, the presence of proteolytic enzyme inhibitors had been detected in plants, although these had been identified in animals during the nineteenth century. Read and Haas (1938) reported that an aqueous extract of soybean flour inhibited the ability of trypsin to liquefy gelatin. The fraction of soybean protein responsible for this effect was partially purified by Bowman (1944) and Birk (1961) and subsequently isolated in crystalline form by Kunitz (1945). Proteolytic enzyme inhibitors are widespread throughout all living entities (Bhattacharyya et aI., 2007). Proteinase inhibitors in plants could be a form of storage protein (Valueva and Mosolov, 1999) or may be to protect the plant against infections and disease. Generally, these proteins form part of a defense mechanism in plants to defend them against proteinases ofpests and pathogens and discourage herbivores (Tiffin and Gaut, 2001; Tamhane et aI., 2005). Proteinase inhibitors cause amino acid deficiencies which affect the development and growth of an insect. When gut proteinases are inhibited or digestive enzymes are vastly over-produced, the insect may die because 12 essential amino acids not available for the synthesis of other proteins (Balter and Jongsrna, 1997; Pompermayer et aI., 2001). Kowalska et al (2007) commented that an abundant source of protein proteinase inhibitors may be found in plant seeds and storage. Other substances, such as tannins (Price and Butler, 1980; Quesada et al., 1996) and several indigestible polysaccharides (Price et al. 1988), may also inhibit digestive enzymes. However, the contribution of these compounds to the total trypsin inhibitor activity (TIA) is minimal in most foods (lkeda and Kusano, 1983). Trypsin is one of the serine-protease inhibitors identified and characterized in plants (Tremacoldi and Pascholati, 2002; Richardson, 1991). In general, trypsin inhibitors are low molecular weight proteins formed by association of identical peptide chains of smaller size. Normally these chains are devoid of carbohydrates, but have between one and eight disulphide bonds (Mueller and Weder, 1990). A common characteristic of plant protease inhibitors is their resistence to pH extremes, heat and hydrolysis by proteases as a result of the level ofS-S bridges (Proteinase inhibitors, 1999). Based on the molecular masses, cysteine content and number of reactive sites, these inhibitors have been categorized into two families, the Kunitz Type and the Bowman Birk Type. Bhattacharyya (2007) identified Bowman Birk Type iubibitors as " ... usually 8-kDa proteins with seven disulfide bridges ... " and Kunitz Type as " ... 20-kDa proteins with just two disulfide linkages". Although active sites are much the same, it would appear that protease inhibitors from different sources have varied structores, molecular weights and chemical compositious (Richardson, 1977; Liener, 1994). These inhibitors inhibit trypsin and chymotrypsin at independent binding sites by reacting with some functional groups in the active site of the enzyme. This inlnbition prevents the substrate from entering the active site or leaves the site catalytically inactive (Bender and Hezdy, 1965; Rittschof et aI., 1990). 13 A protein which inactivates trypsin is found in raw soybeans. This protein causes an increase in the size of the pancreas (hypertrophy), together with increased secretory pancreatic activity (Pimeutel et al., (1996). Lyman and co-workers (Green and Lyman, 1972; Lyman et aI., 1974) have shown that levels of the proteolytic enzymes trypsin and chymotrypsin in the small intestine determine the efficacy of the system regulating negative feedback inhibition, which, in turn, controls pancreatic secretion. Liener (1981) commeuted that the pancreas is stimulated to produce more enzymes when the level of trypsin is reduced: that is, when it is combined with the inlnbitor. Cholecystokinin (CCK) is the major hormone responsible for activating pancreatic enzymes. If too little trypsin is available, CCK is released from the jejunal eudocrine cells of the small intestine (Liddle, 2007). These relationships are illustrated in Figure AI-4. Trypsinogeu .4-------- CCK Trypsin Dietary protein (intestine) Proteolysis Trypsin-Trypsin Inhibitor Figure AI-4: Mode of action of soybean trypsin inhibitors on the pancreas (Anderson et al.,1979) Proteinase inhibitors may add to their natural biological functions, the treatment of human pathologies such as blood clotting and haemorrhage, inflammation and cancer (Neuhof et aI., 2003; Fook et al. 2005; Meno et aI., 2006). 14 A.l.4.2 Amylase inhibitors In order to assimilate starches, which are complex storage carbohydrates, amylase (a digestive enzyme) and other enzymes have to break them down (Choudhury et aI., 1996). Through amylase [a-l,4-glncan-4-glucanhydrolase - Enzyme Code (EC) number 3.2.1.1], starch is broken down to maltose units, then to glucose monomer units. There are plants which have proteinaceous a-amylase inhibitors, as well as proteinase inhibitors (Fraoco et aI., 2000; Mello et al., 2002; Payan, 2004). Several types of plants, especially those in the legume family, have been used to extract amylase inhibitors (Marshall and Lauda, 2006). A large number of sweet potato genotypes have also been reported to contain a-amylase inhibitors (Sasikirao et al., 1999; Rekha et al., 1999). Pancreatic and salivary amylase action is affected by amylase inhibitors (Saunders, 1975; Pace et al., 1978) and faeces reflect this by evidencing a greater proportion of undigested starch. Pusztai et al. (1995) observed that the nutritional quality of the food ingested consequently decreases, and Boivin (1987) surmised that undigested starch in the colon because of high level of amylase inhibitors may cause diarrhea. Diabetes mellitus is a chronic disease, associated with abnormally high levels of glucose in the blood, caused by an insufficient insulin production or lack of responsiveness to insulin or both (WHO, 1999). A means of treating diabetes is through the reduction of postpraodial hyperglycemia by activating decreased absorption of glucose in the digestive tract. The action of carbohydrate-hydrolyzing enzymes, a-amylase and a glycosidase is inhibited to achieve this state, thus decreasing and delaying digestion of carbohydrates. This leads to a reduction in the rate of glucose absorption and a consequent decrease in plasma glucose after meals (Rhabasa-Lhoret and Chiasson, 2004; Ali et al., 2006). It has been claimed that a-amylase inlubitors assist weight loss, although initial research showed them ineffective in reducing carbohydrate absorption (Bo-Linn et al., 1982; 15 A.l.4.3 Hollenbeck et al., 1983; Carlson et al., 1983). Interestingly, subsequent investigation indicated that highly concentrated types of amylase inlubitors could potentially reduce such absorption in humans (Udani and Singh, 2007). Franco et al. (2000) noted that many studies have been undertaken to determine lhe value of a-amylase inhibitors as a means of strengthening a diverse range ofplants against insect and microbial pests. Lectins Lectins or phytohemagglutinins may be defined as large protein or glycoprotein molecules (Liener, 2000) and have been identified in more 1han 800 plant species, including legume seeds, pulses and tubers (Noman et al., 2007; Van Nevel et al., 1998; Ignacimulhu et al., 2000). Molecules which are found on lhe epilhelial cells of the intestinal walls and contain carbohydrate can be bound by lectins. The toxic potential of lectins is determined by lhe degree of lhis binding (Liener,2000). These proteins reflect lheir toxicity by inhibiting growlh in experimental animals (Liener, 1986) and by causing diarrhea, nausea, bloating and vomiting in humans (Liener, 1982). Because lhe presence of lectins irritates lhe intestinal wall., over-secretion of mucus from this membrane may affect its enzymatic and absorptive efficacy. When lectins are together wilh olher anti nutrients, this harmful effect may be more pronounced (Francis et al., 2001). Lectins are classified into several groups based on lheir sugar-binding specificity. Francesca et al. (200I) observed that apparently most lectins have relative masses of between 100 000 and 150 000 daltons and are composed of tetramers. Lectins have the ability to recognize carbohydrates which bind and agglutinate red blood cells and can therefore be used for blood typing (Pusztia, 1992). Autoclaving and aqueous heat treatment - 100?C for 10 minutes - are two melhods which eliminate lectins (Grant, 1991). Thus, there would appear to be little cause for concern regarding toxicity if food is cooked properly. 16 A.l.4.4 Total polyphenols Polyphenols are a large and varied class of metabolites widely spread throughout the plant kingdom: they are a complex but important group of naturally occurring compounds (Ryan et aI, 1999). PhenoIics are secondary metabolites: that is, they are not directly involved in any metabolic process (FAO, 1995). Plants produce them during natura1 development (Harllorne, 1982) and as a result of stress conditions such as wounding, infection and ultraviolet radiation (Beckman, 2000). These compounds, derived from phenylalanine and tyrosine, are an extremely diversified group ofphytochemicals (Shahidi and Naczk, 2004). This class ofplant metabolites contains more than 8000 known compounds, ranging form simple phenols - such as phenol itself through to materials of complex and variable composition, such as tannins (Harllorne, 1993; Bravo, 1998). One thing that all phenolic compounds have in common is that their molecular structme includes an aromatic hydrocarbon group to which a hydroxyl fimctional group ( OH) is attached. Plant foods mostly contain phenolic acids in the bound form. The principal phenolic acids which may be found in plants are alternative derivatives of hydroxybenzoic and hydroxycinnamic acids, the most common of which are hydroxycinnamic acids (Mattila and Hellstrom, 2007). Examples of the latter are caffeic, p-coumaric and ferolic acids, which are often found in foods as simple esters with quinic acid or glucose. These derivatives differ in the patterns of the hydroxylations and methoxylations of their aromatic rings (Shahidi and Naczk, 1995; Hermann, 1989). The methoxylations of their pungent rings and the patterns of the hydroxylations differentiate these derivatives (Shahidi and Naczk, 1995; Hermann, 1989). Phenolics may add to the smen, taste (bitterness, sharpness), colour and oxidative stability of food (Maga, 1978; Robbins, 2003). The dissociability of their -OH group renders phenols acidic. They are also easily oxidized and form polymers (dark aggregates). Phenolic compounds discolour and darken because of enzyme activity, as 17 polyphenoloxidase catalyzes into quinines. As phenolic compounds oxidize, for example, yam tubers which have been cut and left open to the air turn brown (Ozo et al., 1984; Osagie and Opoku, 1984). Plant phenolic compounds are a large and heterogenous group of secondary metabolites with low molecular weight Examples of these are flavonoids, phenolic acids and lignans (Makoi and Ndakidemi, 2007). Phenolic compounds, especially tannins, inlnbit digestive enzymes and interfere with the way in which vitamins and minerals are used. These compounds may also bind and precipitate proteins, thus causing a reduction in nutritional value (Chung et al., 1998; Chavan et al., 2001). Indeed, polyphenols have long been labelled anti-nutrients because of this ability. The anti-nutritional tag may also be a result of the way in which polyphenols inhibit the rate of activity of digestive enzymatic processes. Many enzymes are thus inhibited: for example, hydrolases, protein phosphokinases, isomerases and oxygenases (Ferguson, 2001). Additionally, phenolic acids have strong antioxidant activities and exhibit anticarcinogenic, antiviral, anti-inflammatory, antibacterial and vasodilatory actions (Duthie et aI., 2000; Breinholt, 1999). Flavonoids are polyphenolic secondary metabolites that are omnipresent in higher plants. Many are simply recognized as flower pigments in most angiosperm families (Harborne, 1994). However, their presence is not confined to flowers but include all parts of the plants. About 2/3 of the polyphenols we obtain in our diets are flavonoids. Armstrong (2007) defined flavonoids as: ~ ... 3-ring phenolic compounds consisting of a double ring attached by a single bond to a third ring". 18 HO OH HO 0 Figure Al-S: Structure of a flavonoid (flavonol): Phenolic compound composed of the benzene rings with hydroxyl (OH) groups (adapted from Armstrong, 2007). Many of the flavonoids (Figure AI-S) that occur in plants are in the form of glycosides, where one or more simple sugar, such as glucose or galactose, is attached to them (De Rijke et al., 2006). To sustain healthy tissues and achieve the correct balance of hormones and antioxidants in the body, flavonoids (bioflavonoids and isoflavones) are recommended by some nutritionists. (Brown et al., 1998). However, because flavonoids may precipitate proteins (Jende-Strid, 1991), they demonstrate anti-nutritional properties. Tannins form another group of phenolic compounds, usually divided into hydrolysable tannins and condensed tannins (proanthocyanidins), and are caustic and bitter-tasting. They bind and precipitate proteins, decreasing the digestIbility of protein and carbohydrate. This results in growth depression, in all probablility owing to enzyme resistant substrates formed by interaction between tannins and protein/starch. Digestibility of the substrates is compromised by interaction between tannin and the enzymes (Deshpande and Salunke, 1982; Griffiths, 1986). Deshpande et at. (1986) reported that polyphenols react with proteins and enzymes, so that they can also act as trypsin and amylase inlnbitors. Other anti-nutritioual effects that have been attnbuted to tannins include damage to the intestinal mucosa (Mi~avila et al., 19 A.I.4.S 1977), (Hagerman and Butler, 1991) and an interference with the absorption of iron (Garcia-Lopez et al., 1990), glucose (Welch et aI., 1989) and vitamin B (Singh, 2004). Alkaloids Alkaloids are one of the largest groups of chemical compounds synthesised by plants (Raffauf, 1996). They are generally found as salts of plant acids such as oxalic, malic, tartaric or citric acid (Watkins et aI., 2004) Alkaloids are small organic molecules, common to about 15 to 20 per cent of all vascular plants, usually comprising several carbon rings with side chains, one or more of the carbon atoms being replaced by a nitrogen. They are synthesized by plants from amino acids. Decarboxylation of amino acids produces amines which react with amine oxides to form aldehydes. The characteristic heterocyclic ring in alkaloids is formed from Mannich-type condensation from aldehyde and amine groups (Hendricks and Bailey, 1989). The chemical type of their nitrogen ring offers the means by which alkaloids are subclassified: for example, glycoalkaloids (the aglycone portion) glycosylated with a carbohydrate moiety. They are formed as metabolic by-products. Insects and hervibores are usual1y repulsed by the potential toxicity and bitter taste of alkaloids (Levin, 1976; Robbers et aI1996). Tubers of the common potato (Solanum tuberosum) have a natural content of the two toxic and bitter glycoalkaloids (GA) a-solanine and a-chaconine (Finotti et al., 2006) [Figure AI-6]. The levels are normally low and without adverse affects on food safety and culinary quality. However, consumption of potato tubers with unusual1y high GA contents (300-800 mg kg-I) has occasionally been associated with acute poisoning, including gastro-intestinal and neurological disturbances, in man (Van Gelder, 1991). Tuber GA levels are inheritable and can vary considerably between different species. Environmental factors experienced by tubers during germination, growth, harvest and storage may affect GA levels further (Jadhav et al., 1981; de la Cuadra et al., 1994). 20 0- Sotanine RO II-O-Glucoae~ ? 11-0- Galaclose ...l=1. Q-L.-Rhamno?? ~. a- Ch.conine a-L.-Rhamnose ~ I!-O-Glucose -l=.L ~ Figure AI-6: Structures of two major g1ycoalkaloids found in potatoes: a-solanine and a-cbaconine (Wong, 1989) Alkaloids are considered to be anti-nutrients because of their action on the nervous system (Cheeke and Kelly, 1989), disrupting or inappropriately augmenting electrochemical transmission. Indeed, the physiological effects of alkaloids have on humans are very evident. Cholinesterase is greatly inhibited by glycoalkaloids, which also cause symptoms of neurological disorder (Jackson, 1991). Other toxic action includes disruption of the cell membrane in the gastrointestinal tract (Friedman et aI., 2003). Alkaline pH conditions generally enhance absorption of glycoalkaloids, where binding with sterols in cell membranes causes extra disruption (Roddick, 1979). Lethal doses for humans range between 3 and 6 mglkg body weight, although susceptIbility varies considerably among individuals. A dose of more than 2 mglkg is usually considered toxic 21 (Wong, 1989). Korpan et al. (2004) identified poison symptoms as including vomiting, dianhoea, abdominal pain, apathy, weakness and unconsciousness Nicotine, caffeine, quinine and strychnine are well-known examples of alkaloids. Randolph (2008) claimed that alkaloids in food might well be at least partially responsible for the food-allergy effect of addition, where withdrawal from the food causes disagreeable symptoms. A.I.4.6 Oxalate The plant oxalis, commonly known as wood sorrel, gave rise to oxalic acid (chemical formula HOOC-COOH), a strong, organic acid which has been found to be widely distributed in plants (Liebman, 2002). See Figure AI-7 for structure. OH ~O O~ v---...,-I, HO Figure AI-7: Structure of oxalic acid Strong bonds are formed between oxalic acid and various other minerals, such as calcium, magnesium, sodium and potassium (Fink, 1991; Noonan and Savage, 1999). This chemical combination results in the formation of oxalate salts. A salt formed from oxalic acid is known as an oxalate: for example, calcium oxalate. Some oxalate salts, such as sodium and potassium, are soluble, whereas calcium oxalate salts are basically insoluble. The insoluble calcium oxalate has the tendency to precipitate (or solidify) in the kidneys or in the urinary tract, thus forming sharp-edged calcium oxalate crystals 22 A.1.4.7 when the levels are high enough (Bradbury and Nixon, 1998). These crystals play a role to the formation ofkidney stones (Noonan and Savage, 1999). When oxalic acid is consumed, it irritates the lining of the gut and can prove fatal in large doses. Hui (1992) stated that intake of5g or more ofoxalic acid could be fatal to humans while Munro and Bassir (1969) estimated the threshold ofoxalate toxicity in man to be 2 5g1100g of the sample. Oxalic acid is a common and wide-spread component of most plant families. While the levels of this acid in these plants are generally low, it is the high concentrations in the leaves and conus ofplants consumed daily that are of concern. Oxalate is an anti-nutrient which under normal conditions is confined to separate compartments. However, when it is processed and/or digested, it comes into contact with the nutrients in the gastrointestinal tract (Kaushaiya et al., 1988). When released, oxalic acid binds with nutrients, rendering them inaccessible to the body. If food with excessive amounts of oxalic acid is consumed regularly, nutritional deficiencies are likely to occur, as well as severe irritation to the lining of the gut. Most taro cultivars have an astringent taste and can cause swelling of lips, mouth and throat if eaten unprocessed. This causticity is caused by closely-packed, needle-like, calcium oxalate crystals, which can penetrate soft skin (Bradbury and Nixon, 1998). Thereafter, an irritant, probably a protease, present on the sheathlike bundle of needles (raphides) can cause discomfon in the tissue (Bradbury and Nixon, 1998; Paul et al., 1999). Both the tubers and the leaves can give this reaction (FAO, 1992) but this effect is reduced by cooking (Bradbury and Nixon, 1998). Phytate Phytic acid, which is hexaphosphate of myo-inositol, is very common in the plant kingdom and is found mainly in mature seeds such as legumes, fruits, vegetables and cereal grains (Chan et aI., 2007). Phytic acid is the primary storage compound of phosphorus in plants, accounting for up to 80 per cent of the total phosphorus (Raboy, 2001; Steiner et aI., 2007). Josefsen et aI., (2007) reponed that the negatively charged 23 phosphate in phytic acid strongly binds to metallic cations (e.g. Ca, Fe, K, Mg, Mn and Zn) and forms a mixed salt called phytin or phytate. Phytate has been classified as an anti-nutritional fuctor in the diets of humans. The anti nutritive effect of phytic acid is based on its molecular structure (Figure AI-8). Phytic acid's twelve negative charges from the six phosphate groups bind different di- and trivalent cations into a stable complex. Complete dissociation of phytic acid depends on the pH conditions (pallauf et aI., 1998). Phytate forms insoluble complexes because they are negatively charged under physiological conditions. These complexes cannot be digested or absorbed in the gastrointestinal tract owing to the absence of the intestinal phytase enzyme (lqbal et al., 1994). Deficiencies of phosphorus and nutritionally important minerals in human populations can be a result of cations bound in the phytic acid salt and oflow bioavailability ofphosphorus (Gibson et al., 2006). oI _ O=P-O I QH I : -000- + + OH ,.ca. OH I ./ .??.. I O=P-O' ''0-1'=0 I I o 0 H +. O' _..ca. Itr "O-C-'CHrProtein O=P-O' I o H H Starch IProtein I CH2 .'N.~~_ _ I -,-o-p=O I H er Starch Figure AI-8: Possible interactions of phytic acid with minerals, proteins and starch (Thompson, 1988). 24 The ability of phytic acid to complex with proteins and particularly with minerals has been a subject of investigation for several reasons, predominantly from chemical and nutritional viewpoints. Phytic acid forms complexes with nutritionally important minerals such as calcium, copper and iron (Fez+ and Fe3"). This reaction decreases the solubility of the metals, which are therefore not readily absorbed from the intestine (Brune et aI., 1989; Sandberg etal., 1993; Weaver and Kannan, 2002). There is strong concern that reduced mineral absorption, owing to consumption of food products rich in phytates, may lead to borderline malnutrition (Reddy and Pierson, 1994). It has also been shown that calcium ions interact with protein and phytate to decrease the solubility of proteins further (De Rham and Jost, 1979; Frokiaer et aI., 2001). As a consequence, phytate has been shown to inhibit the proteolysis of a number of enzymes (including pepsin, trypsin and amylases of the intestinal tract) important in digestion (Vaintraub andBlIlmaga, 1991). Certain functional properties of proteins, which are dependant on their solubility and hydration, can be negatively affected by the reduced solubility of proteins as a result of protein-phytate complex. Such hydrodynamic properties (viscosity, gelation) include emulsifying capacity, foaming and foam performance, and dispersibility in aqueous media (Urbano et al., 2000). Phytic acids may also affect the digestibility of starch. Phytic acid and starches are structurally capable of combining via phosphate linkages (Sirkka, 1997). Thus, adverse effects on the digestion of starches and proteins, as well as reduced bioavailability of essential dietary minerals, are the result of high phytate contents in tubers. Investigators have shown that phytic acid may be beneficial with regard to human health, including as an anti-cancer agent (soft tissue, colon, metastatic lung cancer, mammary cancers) [Shamsuddin et al., 1997; Febles et al., 2002], as an inhibitor in renal stone development (Grases et aI., 2000) and as an anti-oxidant agent. Phytic acid has an anti oxidative function because it chelates with iron by combining with all the available Fe 25 A.1.4.8 coordination sides to inhibit OH radical production (Obata, 2003; Muraoka and Miura. 2004). Indeed, it is possible to use phytic acid as an uncommon, versatile food preservative because of its anti-oxidant or iron-chelating properties (Hix et al. 1997). Thompson (1988) suggested that the interaction among phytate, dietary starch and protein could be beneficially utilized in the treatment of diabetes and hyperglycemia. This apparent discrepancy between unhealthy and healthy properties of phytate clearly calls for a re evaluation of this storage compound (Grases et al. 2001). Cyanogens Cyanogen molecules consist of two CN groups that are bonded together at their carbon atoms: N=C-N=C. Cyanogens are glycosides of 2-hydroxynitriles aod are widely distnbuted among plants (Rosenthal aod Berenbaum, 1991; Bisby et al., 1994). When plaots are wounded by herbivores or other organisms, the cellular compartmentation breaks down and cyaoogenic glycosides come into contact with an active l3-glucosidase, which hydrolyses them to yield 2-hydroxynitrile (Vetter, 2000). This is further cleaved into the corresponding aldehyde or ketone aod hydrogen cyanide (HeN) by hydroxynitrile lyase (eq. AI-I) (Conn, 1981). It is the low pH in the stomach which deactivates the 13 glucosidase. Because the environment of the gut is alkaline, reactivation of part of the enzyme fraction is a possibility. The collective name given to glycosides, cyaoohydrins aod hydrogen cyanide is cyanogens (M0l1er and Seigler, 1999). 26 Amygdalin a-Glucosidase Cyanohydrin + 2Glucose i1Hydroxynitrile lyase HCN + C~sCHO Hydrocyanic acid Benzaldehyde Equation AI-I: Breakdown ofcyanogenic g1ycosides to release hydrogen cyanide (Wong, 1989) The ability of plants to produce HCN, known as cyanogenesis, is exlubited by at least 1000 species representing approximately 90 families and at least 250 genera (Miller et al., 2006). About 60 cyanogenic glycosides are known from higher plants (Nahrstedt, 1987; Seigler, 1991) and occur in at least 2000 pIant species, of which a number of species are used as food in some areas of the world. Two examples of staple foods containing cyanogenic glycosides are cassava and sorghum (Okafor, 2004; Agbor-Egbe and Mbome, 2006). Figure Al.9 shows the structure of some of the best known cyanogenic glycosides. Figure Al-9: Structures of some common cyanogenic g1ycosides (A) Linamarin (B) Dhurrin (C) Amygdalin. (Simeonova and Fishbein, 2004) 27 Cytochrome oxidase, the terminal oxidase of aerobic organisms, is the primary site of action for ingested cyanide, an effective inhibitor of many metaIloenzymes (Enneking and Wink, 2000). The enzyme cytochrome oxidase in the mitochondria of cells is inactivated by hydrogen cyanide binding to the Fe2+J Fe3+ contained in the enzyme. This results in a reduction of oxygen usage in the tissues (Vetter, 2000) and oxygen starvation at cellular level, owing to the effects of cyanide poisoning, can result in death. Respiratory failure is therefore the cause of death since the respiratory centre nerve cells are extremely sensitive to hypoxia (Okolie and Osagie, 1999). Other long-term diseases associated with dietary cyanide intake include (i) konzo (Cliff et al., 1997), a paralytic disease; (ii) tropical ataxic neuropathy (TAN) [Onabolu et al., 2001], a nerve-damaging disorder that renders a person unsteady and uncoordinated; (iii) goiter and cretinism (Delange et al., 1994). More than 10 000 people in Mozambique, Tanzania and the Democratic Republic of the Congo have been paralysed by a devastating disease that developed from prolonged exposure to sublethal levels of dietary cyanide (Shorter, 1997). Sheeba and Padkaja (1997) observed that the palatability and shelf-life of cassava products may be prolonged by processing. The levels of cyanogenic glycosides and hydrogen cyanide are also reduced to safer limits by processing (peeling, slicing, boiling) before consumption (Sheeba and Padmaja, 1997; Feng et al., 2003). Bourdoux et af., (1982) and others concluded that, based on total root cyanide content, less than 50 ppm may be classified as innocuous; 50-100 ppm as moderately poisonous; and more than 100 ppm as dangerously poisonous. In 1991, the World Health Organisation (WHO) set the safe level of cyanogens in cassava flour at lOppm (FAOIWHO, 1991). The acceptable limit in Indonesia has been set at 40 ppm (Damardjati et al., 1993; Djazuli and Bradbury, 1999). 28 A.I.4.9 Saponins Saponins (Figure AI-IO) are a group of varied, biologically active glycosides which derive their name from their ability to form stable, soaplike foams in aqueons solutions. They represent a complex and chemically diverse group of compounds. Saponins consist of a polycyclic aglycone that is either a triterpenoid (C30) or steroid (C27) sapogenin, attached via ester and ether linkages to a sugar side chain (Schwarz, 1993). Glucose, galactose or a pentose or methylpentose may make up the sugar moieties. Me HO HOCHz 0 .:ko~ Me: H004 6~H OH Figure AI-IO: Molecular structure of saikosaponins from bupleurum. (Dharmananda, 2000) Saponins have purgative properties, forming oil-in-water emulsions and producing abundant amounts of foam when dissolved in water. The ability of a saponin to foam is caused by the combined hydrophobic sapogenin and the hydrophilic side chain. Saponins are non-volatile compounds that are known to have a bitter taste and reduce the palatability oflivestock feeds (Curl et al. 1985; Bishnoi and Khetarpaul, 1994). It is this undesirable, bitter taste that creates a major problem in the utilization of saponin containing plants and their products (Davies and Lightowler, 1998). 29 However, if saponins have a triterpenoid aglycone with glucuronic acid, they may taste of licorice, sweetened by the sugar moiety (Grenby, 1991). Saponins occur in a wide variety ofplants such as peanuts, lentils, lupins, alfalfa, soybeans, peas, oats and spinach (Smartt, 1976; 0akenfulL 1981; Price et al. 1987; Cuadrado et al., 1995; Hubman and Sumner, 2002). Saponins are attracting considerable interest as a result of their diverse properties, both deleterious and beneficial (Cuadrado et al., 1995). One of the well-known biological effects of saponins is their ability to split erythrocytes (Khalil and EI-Adaway, 1994) and to make the intestinal mucous membrane permeable (Johnson et aI., 1986). Saponins are amphiphilous compounds which interact with biomembranes of animals, fungi and even bacteria. The hydrophobic part of the molecule forms a complex with cholesterol inside the membrane and their hydrophilic sugar side chain binds to external membrane proteins. Thus, the fluidity ofbiomembranes is disturbed, which leads to the formation of holes and pores. As a consequence, cells become leaky and die (Oakst and Knowles, 1977). Clinical reports have also shown that the immune system is affected by saponins in ways that lower cholesterol levels and help to protect the human body against cancers (pathirana et al., 1980). This hypocholesterolemic effect has been ascribed to a complexation of the saponin with cholesterol and bile acids, which results in a decrease in their absorption from the intestines (Matsuura, 200I). A saponin-rich diet can be used for the treatment of hypercalcinria in humans, as an antidote against lead poisoning and in the inlnbition of dental caries and platelet aggregation (Shi et al. 2004). Saponins also reduce blood lipids (Chunmei et al., 2006), lower the risks of developing cancer (Messina and Bennik, 1998) and lower blood-glucose response (Sajadi Tabassi et al. 2007). A.I.S Effect ofproeessing on nutrients and anti-nutrients Several fuctors influence the nutritional content of food. These include the genetic make-up of the plant, the soil in which it is grown, nse of fertilizer, prevailing weather, maturity at 30 .- harvest, packaging, storage conditions and melhod utilized for processing (Morris et al., 2004). The primary purpose ofprocessing is to render food palatable and develop its aroma, but it has inevitable consequences on lhe nutritional value of foods. Washing and peeling result in lhe loss ofmany water-soluble vitamins, since lhese are more concentrated in lhe peel and outer layers. Wilh careful control of the processes, nutrient losses can be minimized wilhout affecting palatability. The term "food processing" covers an enormous field, from simple boiling to lhe use of irradiation. The types of cooking melhods differ in countries around lhe world and also vary wilh lhe elhnic background of lhe family. Processing (cooking) can be bolh beneficial and detrimental to nutrient composition of foods. It is known that processing techniques may decrease the food value of some nutrients (Nestares et al., 1996): for example, lhere is some inevitable leaching of nutrients into lhe cooking water during processing. The cooking water may or may not be discarded, depending upon cultural and personal preference. On lhe olher hand, processing may enhance the nutritional quality of food by reducing or destroying the anti-nutrients present in it, as well as increasing the digestibility of proteius and starches. Elimination or inactivation of anti-nutritional compounds is absolutely necessary to improve lhe nutritional quality and effectively utilize human foods to lheir full potential. Processing generally inactivates heat-seusitive factors such as enzyme inlnbitors, lectius and volatile compounds such as HCN. A typical example is lhe protein in legumes, which is made more digestIble by heating because of the inactivation of anti-nutrients such as trypsin inlnbitors (Siddhuraju and Becker, 1992). The use of some processing methods, such as boiling, baking, microwave- and pressure cooking are known to achieve reduction or elimination of anti-nutritional factors (Udensi et aI., 2005; Bhandari and Kawabata, 2006; Habiba, 2002; Khokhar and Chauhan, 1986). Radiation processing and extrusion have also been used as a means to inactivate anti- 31 A.l.6 nutritional factors naturally present (EI-Niely, 2007; Alonso et al. 2000). Extrusion cooking was found to be a more versatile, quick and efficient method to reduce anti nutrients when compared with other traditional processing methods (Alonso et al. 2000). Soaking, sprouting, fermentation and cooking methods have also been investigated. Combination of cooking and fermentation improved nutrient quality and drastically reduced the anti-nutiritional factors to safe levels much greater than any of the other processing methods tested (Obizoba, 1991). Excessive heat processing, however, should be avoided, since it adversely affects the protein quality of foods. It is therefore important that processing is done within the recommended guidelines e.g. for heat, pH, as over processing will further destroy not only nutrient content but also taste and appearance (Morris et aI., 2004). Aim and outline of Section A Taro (Amadumbe) is a staple food extensively eaten in the tropics and subtropics of Africa (Oscarsson and Savage, 2007). The nutritional value of taro has been extensively studied, but the researcher is not aware of any such study pertaining to Amadumbe, the South-African cultivar: hence, the importance of investigating the nutritional quality of Amadumbe. Different domestic processing methods are used to reduce the heahh risk associated with food consumption. It is therefore necessary to estimate the levels of nutrients and anti nutrients in processed and unprocessed Amadumbe. The objectives of Section A were: ? to evaluate the chemical composition of three Amadumbe phenotypes (proximate, mineral and anti-nutritional factors) among these phenotypes; ? to investigate the effect ofdomestic processing methods (boiling, frying, roasting) on anti- nutritional factors present in Amadumbe. 32 CHAPTERA-2 MATERIALS AND METHODS A.2.1 Introduction This chapter gives a brief description of the materials and methods used to determine the nutritional and anti-nutritional composition of Colocasia esculenta grown in Zululand, South Africa. See Appendices A and B for details. A.2.2 A.2.2.1 A.2.2.2 Materials Food material Two varieties of Colocasia esculenta L. Schott (Amadumbe) - white and purple - were obtained from local markets at Esikhawini and Mtubatuba and from Makatini Experimental Farm, KwazuIu-Natal (KZN), South Africa Reagents (See Appendix A for details ofhow reagents were prepared.) a-Amylase a-Chymotrypsin from human pancreas l3-glycosidase Ammonium molybdate Anthrone reagent Ascorbic acid Benzoyl-DL-arginine-p-nitroaniline (BAPNA) Bispyrazolone 33 Sigma Sigma Sigma Kleber chemicals Merck Merck Sigma Fluka Bromocresol green Calcium oxalate Dinitrosalicylic acid (DNS) Ferric ammonium sulphate [Fe~(S04h] Papain Phosphoric Acid (H3P04) Polyvinylpolypyrrolidone (pVP) Potassium antimonytartrase Potassium cyanide (KeN) Potassium ferricyanide [K3Fe(~1 Potassium hydrogen phosphate (K.2HP04-3H20) Potassium permanganate (KMn04) Protease: Bacillus Licherniformis Pyridine Saponin Sodium phosphate (Na3P04) Trypsin Trypsin type I Bovine pancreas Tungstophosphoric acid Type xiii Fungal protease: Aspergillus saitoi Type xiv Bacterial protease: Streptomyces griseus Type xviii Fungal protease:Rhizopus Vanillin 34 Sigma-Aldrich Fluka Merck Merck Sigma Merck Fluka Merck Merck Merck Merck Merck Sigma Merck Sigma Merck Sigma Sigma Merck Sigma Sigma Sigma UNILAB A.2.2.3 A.2.3 A.2.3.1 A.2.3.2 Special equipment utilized Soxhlet Extractor (Soxtec HT-6, Tacater AB, Hoganas) PerkinElmer Atomic Absorption Spectrophotometer (Carl Zeiss) Model AAS-3 GPR centrifuge (Beckman) Spectrophotometer (Pharmacia biotech-novaspec) DV Spectrophotometer Laborota 400 Rotary evaporator (Heidolph) Methods (See Appendix B for details ofmethodology) Sample preparation Healthy Amadumbe tubers were washed, hand-peeled and cut into small pieces. Only tubers with little or no skin wounding were selected. Samples were divided into two groups: one group was referred to as processed and the other group as unprocessed. The unprocessed samples were dried at 55? C for 24 hours and then were milled into a fine powder which was stored in brown bottles until used. The processed portion was subdivided into three different parts (those to be boiled, fried and roasted), processed as descnbed in Section A.2.3.2 and screened for the presence ofnutrients and anti-nutrients. Processing techuiqoes Approximately 500 g each of the processed portion of Colocasia esculenta samples were separately subjected to the following processing techniques: I. Boiling: Amadumbe samples were boiled in distilled water on a stove for 30 minutes, after which they were drained. ii. Frying: Amadumbe samples were deep-fried in domestic cooking oil (100 per cent sunflower oil) for 15 minutes. 35 A.2.3.3 A.2.3.4 U.3.4.1 iii. Roasting: Amadumbe samples were roasted in a baking pan in an oven for 30 minutes at 1800 C. The processed samples were sun-dried and then milled into a fine powder, which was stored in bottles until used. Proximate composition Protein, moisture, carbohydrate, ash and crude fat contents were determined as descnbed in AOAC methods (1990). The crude protein content was calculated by converting the nitrogen content determined by the micro-Kjeldahl method (N x 6.25). The moisture content was determined by oven-drying at II00 C to a constant weight. The total carbohydrate and starch contents were percolated with ethanol and perchloric acid respectively and then reacted with anthrone (Hansen and Msller, 1975). Ash content was determined by incineration of the samples, the weights of which were known, in a furnace oven. Fat content was determined gravimetrically by extraction, using petroleum ether on a Soxhlet extraction unit. The dried residue was quantified gravimetrically and expressed as percentage of lipid. Fatty acid composition in Amadnmbe Lipid extraction Purple and white Amadumbe samples from Esikhawini local market were separately extracted with a methanol-ehloroform (2:1, v/v) mixture over 24 hours in an oven shaker at room temperature. This was then filtered under suction and the residue re-extracted with a methanol-ehloroform-water (2.5:1.3:1, v/v) mixture for another 24 hours. The filtrates were combined and then separated in the separating funnel into chloroform and water fractions. The chloroform layer was then mixed with 30 ml of benzene and dried under vacuum. The residuallipids were weighed and redissolved in a minimal volume of a methanokhloroform mixture and stored in the fridge (Bligh and Dryer, 1959). 36 A.2.3.4.2 A.2.3.4.3 A2.3.4.4 In another experiment, Amadumbe samples were extracted with hexane. The extract was filtered through rough filter paper and then finely filtered through a 0.45 J.1ID. pore filter paper. The hexane was evaporated in vacuo and the residue dissolved in an acetonitrile-2 propanol-hexane (2:2:1, vlv) mixture. Iodine value A known amount (5 mg) of the methanol-ehloroform extracted lipid was dissolved in chloroform and Dam's reagent was added. The mixture was then left at room temperature in a dark room. Potassium iodide was added as the source of iodine in the mixture with the starch indicator. The mixture was titrated with thiosulfate solution to determine the h1Jerated iodine. The value was used to determine the number of double bonds present in the lipid extract. (Yasuda, 1931; AOAC, 1984). Column chromatography About 20 g of silicic acid was preheated in the oveo at 1200 C. A slurry was then prepared with 35 00 of chloroform and poured into a column chromatography tube (48 nun length x 2.15 mm internal diameter). The air in the tube was gently disloged and the column was washed with 2 column volumes of chloroform. The sample was then runned and eluted with differeot solveots of chloroform (175 00), acetone (700 ml) and methanol (17500). High Pressure Liquid Chromatography-Mass Spectroscopy (HPLC-MS) The hexane extracts of Amadumbe were redissolved in methanol and water (1:1, v/v). The lipid extracts were then injected into a LCMS machine to determine the fatty acid composition. Standard fatty acids were similarly treated. Their profiles were used to ideotify and quantify the fatty acids ofAmadumbe (See Appendix C). 37 A.2.3.5 A.2.3.6 A.2.3.6.1 Mineral analysis The standard Association of Official Analytical Chemists (AOAC, 1990) method was used to digest the samples. The digests were diluted with HCl and the mineral composition and concentrations of Na, Ca, K, Zn, Fe and Mg were determined, using a PerkinElmer Atomic Absorption Spectrophotometer. Determination of anti-nntrients Trypsin inhibitor The method descn1Jed by Smith et al. (1980) was used to determine the antitrypsin activity. Trypsin activity was measured by using BAPNA as substrate in the presence and absence of a sample extract. p-Nitroanilide released was measured using a spectrophotometer at 410 mn. Trypsin inlnbitor activity (TlA) was therefore expressed as the decrease in trypsin activity per unit weight of sample, using the formula: 2.632?D ?At TlA S mg pure trypsin inhibited g-l sample A.2.3.6.2 D is the dilution factor, At is the change in absorbance and S is the amount of sample weighed out. Amylase inhibitor a-Amylase and a-amylase inhibitory activities were estimated according to the method utilized by Bernfeld (1955). One a-amylase unit (1ill) was defined as the amount of enzyme that will liberate IJllllol of maltose from the starch under the assay conditions (10 minutes, 37" C, pH 6.9). The amylase inhibitors activity (AlA) was determined as the percentage decrease in a-amylase activity (at the stated conditions) in the presence of Amadumbe extracts. 38 A.2.3.6.3 \.2.3.6.3.1 \.2.3.6.3.2 Lectin Amadumbe samples were homogenized with a sodium borate buffer m a shaker overnight. The samples were then filtered and serial dilutions were made. CoUection of platelets The rats were anaesthetized with ether and blood was immediately collected from abdominal aorta into centrifuged tube containing ADA (anticoagulant) [1 m! ADA: 5 m! blood]. The blood was then centrifuged (15 minutes at 1200 rpm, 3 minutes at 2200 rpm and 15 minutes 3200 rpm) and resuspended in 5 m! of washing buffer [1 m! sediment: 20 m! resuspending buffer]. Measurement of platelet aggregation The method ofHwang et al. (1974) as modified by Mekhfi et al. (2004) was adopted for the measurement ofplatelet aggregation. A 1:20 dilution of platelets was prepared in RB. A sample of washed platelets (0.4 ml) was mixed with CaCh to a final concentration of 1.3 mM. Aggregation was initiated by adding 1 Ulm! of thrombin. The development of platelet aggregation was recorded at 546 um over five minutes. Experiments with Amadumbe extracts were monitered by pre-incubating the washed platelets with extracts for one minute. The platelet aggregation was immediately initiated by adding tbrombin. Percentage of stimulation of clotting was calculated by using the formula: !:lA control + !:lA test x 100 % stimulation !:lA control !:lA is the change in absorbance at 546um 39 A.2.3.6.4 A.2.3.6.5 A.2.3.6.6 A.2.3.6.7 Total polyphenols The Prussian Blue Method, utilized by Price and Butler (1977), was used to determine total phenol content The phenols were extracted into 2 M HCL Timed additions were performed on the extracted sample, using 0.10 M Fe~(S04)2 and 0.008 M K3Fe(CN)6 to develop colour. Absorbance was measured spectrophotometrically at 720 nm. The total phenol concentration was calculated and expressed as a gallic-acid equivalent Tannin Tannins were determined by the method utilized by Van-Burden and Robinson (1981). The Amadumbe samples were weighed and extracted into distilled water. 0.1 M FeCh in 0.1 N HCl and 0.008 M potassium ferrocyanide were added to the filtrate. The absorbance was measured at 120 nm within 10 minutes. Gallic acid was used as a standard to draw the standard curve, from which the tannin content was estimated. Flavonoid Flavonoids were determined using the method of Boham and Kocipai-Abyazan (1994). Samples were repeatedly extracted into 80% aqueous methanol. The filtrate was evaporated into dryness over a water bath and weighed. The percentage yield of the flavonoid was calculated. Cyanogens Cyanogens were assayed enzymatically, using the method descnbed by O'Brien et al. (1991). Amadumbe samples were homogenized in a 0.1 M orthophosphoric/ethanol extraction medium. An aliquot of extract was added to a phosphate buffer (O.lM H3P04 and Na3P04; pH7) and 13-glycosidase (5EU ml- l ). The lIberated HCN was reacted with pyridinelpyralozone reagent to develop colour and the absorbance was measured spectrophotometrically at 620 nm. KCN was used as a standard to draw the standard curve, from which the cyanogen content was estimated. 40 A.2.3.6.8 A.2.3.6.9 A.2.3.6.10 A.2.3.6.11 Phytate Double acid extraction (HCl and H2S04) was perfonned on sample materials for three hours. Samples were filtered under vacuum through Wbatrnan no 1 filter paper. Colour was developed by adding ammonium molybdate, ascorbic acid and potassium antimonytartrase and absorbance was measured at 820 run. A standard curve was prepared, expressing the results as potassium-hydrogen-phosphate equivalent. The concentration ofphytate was calculated from its phosphorus content. Alkaloid Amadumbe alkaloids were detected by the method utilized by Harbome (1973). Amadumbe samples were soaked in a 10 per cent solution of acetic acid in ethanol for four hours. This was filtered and the extract was concentrated on a water bath. Precipitation was executed by adding concentrated ammonium hydroxide. The precipitate thus obtained was washed with diluted ammonium hydroxide, dried and weighed The percentage yield of the alkaloid was calculated. Oxalate Oxalate was detennined employing the method used by Munro and Bassir (1969). The oxalate was extracted with 0.15 per cent citric acid and treated with tungstophosphoric acid Precipitated oxalate was solubilized with hot diluted H2S04 and titrated against KMn04. Oxalate content was expressed as calcium oxalate equivalent. Saponin Saponin content was detennined utilizing the method employed by Fenwick and Oakenfull (1981). Saponin was extracted for 24 hours in a reflux condenser containing pure acetone. Re-extraction with methanol in the Soxhlet apparatus was carried out for another 24 hours. Colour development was achieved by using vanillin in ethanol and 41 sulphuric acid. Absorbance was measured spectrophotometrically at 500 DIn. Saponin was used as a standard to draw the standard curve, from which the saponin content of sample was estimated. All analyses were done in duplicate and the results reported are the mean values. 42 CHAPTERA-3 RESULTS A.3.1 Introduction Nutritional value is the main concern when a crop is considered as a food source. Amadumbe is cultivated as a subsistence staple in parts of South Africa Information on nutritional and anti-nutritional values of processed and unprocessed Amadumbe tubers grown in KZN, South Africa is given in this chapter. A.3.2. Proximate composition The proximate composition of the two varieties of Colocasia esculenta tuber, from different locations, was determined by standard procedures. The data for the processed and the unprocessed samples are presented in Tables A3-la - A3-lc. In general, the proximate composition of the two varieties of Amadumbe studied is similar to that of all known tubers. However, differences between the two varieties, as well as between the different locations, were observed in the proximate composition values obtained. The moisture and ash contents of Amadumbe are presented in Table A3-la. Water content was high in the investigated starchy staples, which, on average, ranged between 84 and 89 per cent. The unprocessed Esikhawini varieties showed the highest moisture content 43 Table A3-1a: The moisture and ash content (g/100g DM) of processed and unprocessed Amadumbe (Co/ocasia escule1ltJl) tnOOrs Esikhawini white (EW) 89 4.4 Boiled white (BW) 88 4.4 Roasted white (RW) 84 3.6 Fried white (FW) 85 4 Esikhawini purple (EP) 89 3.3 Boiled purple (BP) 88 3.2 Roasted purple (Rp) 87 4 Fried purple (FP) 85 3.2 Mtnbatnba white (MtW) 87 5.4 Mtnbatnba purple (MtP) 86 4.4 Makatini white (MakW) 87 5.4 Makatini purple (MakP) 86 4.9 The ash content of Amadumbe tubers ranged between 3.2 and 5.4 per cent of the dry weight material. The mean ash content for the unprocessed tubers was 4.6 per cent and that of the processed tubers, 3.7 per cent. The ash content of Amadumbe is significant in that it contains nutritionally important minerals. The erode fat and protein content of the Zululand Colocasia esculenta species are shown in Table AJ-Ib. 44 Table A3-1b: The crude fat and crude protein composition (gf lOOg DM) in unprocessed and processed Amadumbe Esikhawini white (EW) 0.8 5.04 Boiled white (BW) 0.78 5.04 Roasted white (RW) 1.58 6.87 Fried white (FW) 10.58 4.2 Esikhawini purple (EP) Boiled pmple (BP) Roasted pmple (RP) Fried pmple (FP) 0.28 4.5 0 4.56 2.52 4.36 15.05 3.95 Mtubatuba white (MtW) Mtubatuba purple (MW) Makatini white (MakW) Makatini purple (MakP) 1.54 1.06 0.73 0.99 5.39 3.72 5.08 3.89 The lipid content for the unprocessed conus ranged between 0.73 and 1.54 per cent. The crude fat content for fried EW and EP Amadumbe tubers (10.58 and 15.05 per cent respectively) was higher than the mean crude fat value for the boiled and roasted samples, as well as higher than that of the unprocessed samples. The crude protein content of the unprocessed Colocasia esculenta tubers was found to range between 3.72 and 5.39 per cent MtP tubers presented the lowest crude protein content, whilst MtW showed the highest values. All the unprocessed tubers had a protein content of less than six per cent. No apparent increases or decreases were observed in the crude protein content for processed tubers. 45 Table A3-1c Carbohydrate content (g% DM) ofnnprocessed and processed Colocasia escu/enta tubers Starch Soluble sugar Esikhawini white (EW) 28 4.0 Boiled white (BW) 25 3.0 Roasted white (RW) 27 2.0 Fried white (FW) 18 2.7 Esikhawini purple (EP) 25 2.0 Boiled purple (BP) 22 3.1 Roasted purple (RP) 20 2.5 Fried purple (FP) 15 2.0 Mtubatuba white (MtW) 16 1.1 Mtubatuba purple (MtP) 19 1.7 Makatini white (MakW) 24 2.3 Makatini purple (MakP) 23 2.1 The unprocessed Colocasia esculenta tubers revealed high levels of starch (28-16 per cent), the predominant component of dry matter (Table A3-1c). The soluble sugar content of Amadumbe tubers ranged from 1.1 (MtW) to 4.0 per cent (EW) on a dry-weight basis. Processing decreased most of the total carbohydrate content of the Amadumbe tubers, poSSibly because of the leaching away of the component. 46 A.3.3 Mineral analysis The composition of mineral nutrients in the processed and unprocessed Amadumbe species studied are given in Tables A3-2a and A3-2b. Table A3-2a: Macronutrient profile ofnnprocessed and processed Cowcasia esculenta (mg/lOOg DM) --Esikhawiniwhite 11.1 24.7?0.26 408 79.6?0.77 ? ? Boiled white Roasted white Fried white 13.9 25.8 23 35.3 ? 0.27 41 ?0.80 32.5 ? 0.59 312 362 350 102.6 ? 1.17 101.5 ? 0.69 103.1 ? 1.37 ? ? ? ? ? ? Esikhawini purple 26.4 40.3 ? 0.53 360 88.9 ? 1.10 ? ? Boiled purple 42 39.4?0.62 250 99.6 ? 2.71 ? ? Roasted purple 17.2 45 ? 1.13 298 103.9 ? 1.22 ? ? Fried purple 27.02 33.5 ? 0.41 268 74.1 ? 0.72 ? ? Mtubatuba white Mtubatuba purple Makatini white Makatini purple 13.4 20 19.6 14.3 41.3 ?0.06 47.8? 1.13 37.5 ? 0.58 41.4 ?0.60 464 458 740 642 108.6 ?0.9l 122.1 ? 0.52 93.45 ?0.77 121.5 ? 1.18 ? ? ? ? ? ? ? ? ? Macro-element (not quantified) present in Amadumbe tubers In general, a variation in mineral distnbution was noted between varieties. The presence of 15 minerals was investigated in the processed and unprocessed tubers; six were quantified (Tables A3-2a and A3-2b). The composition and concentration levels of these nutrients 47 varied significantly among cultivars. Potassium was the most abundant macromineral (740 mg 100 g-I DM) in the unpmcessed tubers. Magnesium was the second most common mineral and varied between 79 and 122 mg 100 g-I DM Appreciable amounts of calcium (24.7- 47.8 -I DM) and sodium (lU - 42 mg 100-1 DM) were also noted. Table A3-2b: Micl'lHllement contents ofunprocessed and processed Co1Dcasill esculentJl (mg/lOOg DM) ~~__~~.t~ Esikhawini white . 2.13 ? 0.03 2.33 ? 0.05 ? ? ? ? ? ? ? Boiled white 3.25 ? 0.05 Roasted white 3.37 ? 0.10 Fried white 3.09 ? 0.08 2.62 ?0.03 4.34 ? 0.02 11.48 ? 0.19 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Esikhawini purple 3.83 ?0.06 2.12 ?0.03 ? ? ? ? ? ? ? Boiled pmple 2.99 ? 0.09 2.58 ?O.06 ? ? ? ? ? ? ? Roasted purple 2.18 ? 0.12 6.65 ?0.20 ? ? ? ? ? ? ? Fried purple 2.45 ?0.05 12.87 ?0.02 ? ? ? ? ? ? ? Mtubatuba white 1.44 ? 0.02 Mtubatuba purple 5.27 ? 0.15 Makatini white 2.49 ? 0.06 Makatini purple 3.17 ? 0.07 4.13 ?0.06 2.07 ?0.02 3.12 ? 0.02 2.29 ?0.002 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Micro-element (not quantified) present in Amadumbe tubers With regard to micronutrients (Table A3-2b), Zn was the most abundant, with 3.05 mg 100 g-I DM as a mean value for the unprocessed tubers. Iron content ranged between 2.07 48 and 4.13 mg 100 g-l DMin the unprocessed tubers. The concentration of iron and zinc was the lowest ofquantified minerals observed in the varieties studied. A.3.4 Fat and fatty-acids composition The percentage yield of crude fat extract from Amadumbe with methanol-chloroform is presented in Table A3-3a Table A3-3a: Yields of crude fat of Amadumbe extracted with methanol-chJorofonn mixture (2:1, vlv) Esikhawini white Esikhawini purple 3.3 3.6 The iodine numbers and the corresponding degree of unsaturation for the extracted lipids are presented in Table A3-3b. Table A3-3b: Iodine value ofAmadumbe lipid extract Esikhawini white Esikhawini purple 73 29 37 15 Figures A3-l (a-d) shows the results of the investigation into the composition of fatty acids in Amadumbe, using HPLC-MS chromatographic techniques. 49 r.=-- I:TOFMSd I' EWM 11 1.1"'IIir 6.411 I 0I 0.00 2.00 ?.00 6.00 6.00 10.00 12.00 1?.00I:TOFMSES- I EWM 7.81 8.91 I I 7J101 11.51l 'UlO 11 0.00 2.00 ..00 lUIO 6.00 10.00 12.00 1??00 EWM 1: TOFMS ES- I 8AO 281 8. 7.81 12..r 0 0.00 2.00 ?.00 6.00 6.00 10.00 12.00 '''00 IEWM ':TOFMSES-72 7.81 UO Il.I3 t.21 U7 0.88 1Q.03 IlSl '.19 4"'''35 L74 5.78 6.411 ,1 { : >:-:-, =do \ ;' TIme0.00 2.00 ??00 8.00 8.00 10.00 12.00 1??00 Figures A3-1 a Esikhawini white Amadumbe extract (methanol-chloroform) 50 IF-- ~ 3.58+1 I 3.08+, ,.... 2._' .q 2.08+' ,._, 1.08+1 5. 'U8 D. D.DD 2.00 4.DD EWM 6.DD 8.00 7.B1 8.40 721 &18 821 lD.DD 111.15 11.32 12.DD 14.DD I:TOFMSES TIC 1.47 os Q.BI 1.19 74 ll1JlS '0.74 14.00 Figures A3-1 al Esikhawini white Amadumbe extract extension (methanol chloroform) 51 1t.IIr- I!EWH: ? ..40 \ 1: TOF lIS m 281 .. 12.IIl 12.li1 7'" 7.81I ~1:-00~~~--2.lJO"""-"T""-~0~.00~-~~~,,~00~-~-'?''''''''1II-.L.'';:'''''-~'0~.oo----~12.IIO~.c>...,.-~10T.oo~""'" EVVH 1: TOF MS Es.- .. 7111 111. 11,.;0 ~oob-~--r--:2.'"'00:::-'~""~-0"-.00c:c----r--:8.'"'00:::-,--<>/-l....L~&lIIC-""'''''~"""''1~Q.':'00:,"",~.-AJ,2.~00"':'''"-......--,~O.,..,III-.--, EWH 1: TOF lIS m- 6.1 .... 0 0.00 2.111 0.00 6.00 0.00 10.00 12.00 ,.00 EWH 1: TOF MS El>-: 7 "lli~ 11C1.-4.3l U7 ..70 Q.I2 ..., .... IU, 11.1':!1Ot2.44Q. .... 1 Tmo 0.00 ----L..-- 2.00 0.00 &.00 ..00 10.00 12.111 14.00 Figures A3-1 b Esikhawini white Amadumbe extract (bexane) 52 fattyacld_ EWi lD.ll5 1.4e+l 1.28+1 loq i EWH 1'-1 8. .... 2.00 ".211 UlI ".00 8.00 8.00 7.21 7.ll1 BAD 1.'8 9.21 10.00 10.7.. lDJl1l 12.00 1".00 1:1OFMSES TIC 1._ 7.. 0JI2 o.n 2.lI5 1 h:..E.~~:::::S;::;:::::.::::'--.-.-';::~~~~~.....,..._~-=:"-'':-;:..'C:::-:'-...:.::;:;::::::::;:;:::-.. 1'Ine ___--MlL. ~2.!!'OOi!.._ ___'4!_.OO!'!_ _'8.~OO!!!!... _'Il~OO!!!!... ...!1~D.!!'OOi!.._ ___'1~2.!!'OO"_ ___'14".!!'OO"_ _ Figures A3-I bI Esikhawini white Amadumbe extract exteusion (hexane) 53 -------r 1:WF:~ I 7.8' 4.32 5.13~!::OO'""-"--'2-:"!00::-~'"T'"~"'4':'.llOE-.:><>..;;'o;;;.,.~8.:'100~~""'::-~I.!'00""-""-""o.:;00:-=:-"F(?';',~2.llOT'" ............-,~4_..1IO-......, EPH 1: WF lIS ES- 7. ? ratIy-- EPH un l1.58,UO ?b~"--"""'-'''''''''--""T'"~'''''''''~''''''~"""':T---f>.,-.l~-~......~.,....,~..,......,4.,L;.,.~....,...~...,.. ..........., 0.110 2-110 4.00 1.110 8.00 10.00 12-00 EPH llAO 1ZA1 0 0.00 2-00 4.00 8.00 '0.00 12-110 14.110 EPH 1: WFMS ES- 8.40 TIC 1. 7.81 1i'- 1121 O.lll 4.211 ~1.ll7 "-.J \f'~Il ''1..ll8 10l'11~ '~ }ZA11 nn. 0.00 2-00 4.00 8.00 8.00 10.00 12-00 1?.00 Figures A3-1 c Esikhawini pnrple Amadnmbe extract (methanol-chloroform) 54 rau,- EPH 'U6 5.0 4.0 u_~ Rqe:7 I 4.00 8.00 8.00 7.111 g. 10.00 Ill.ll11 lU7 12.00 14.00 1: 10F lIS ES-' TIC1._ Figures AJ-l cl Esikhawini white Amadmnbe extract extension (methanol chloroform) 55 i8iiY acid abet -- EPM Le 7.85 .~ 1:TDFMS~ ~1 Figuns A3-1 d Esikhawini pUrple Amadumbe extract (bexane) 56 aIIy- _le 1.2e+2 9All 1.00+2 8.. 4.00+1 2._, 14.00 1: TOF lIS Ell TIC 1. .2.0010.00 1.43 a.. 8.15 Il.lIIl 7.I11i 6.004.002.00 o..oi..-IJ.~~......................==..............,......~......~~-4~~~=='<'==";==F __"" 0.110 o.nO.1I1 EPMLC fS.lID 7.211 '0.75 Figures A3-1 dl Esikhawini purple Amadumbe extract extensiou (hexane) Figures A3-1 (a-d. and al-dl) Chromatographic separation of Amadumbe (ColocasiJl escuIenta) fatty acid Each peak on the separate chromatograms in Figure A3-I (a-d) show the retention time with the relative molecular weights for the lipid extracts. 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Part I: diagnosis and classification of diabetes mellitus. Report ofa WHO Consultation, Geneva Yasuda M (1931) The determination of the iodine number of lipids. J BioI Chem 94, 401-409. Yamori Y., Nara Y., MizJ!shima S., SawamuraM, Horie R. (1994) Nutritional factors for stroke and major cardiovascular diseases: international epidemiological comparison of dietary prevention. Health Rep 6, 22-27. Zhang H., Xie X., Xu Y. and Wu N. (2004) Isolation and functional assessment of a tomato proteinase inhibitor IT gene. Plant Physiology and Biochemistry 42, 437-444. 117 SECTIONB PURIFICATION AND AMYLASE INHIBITOR COLOCASIA ESCULENTA SUMMARY CHARACTERIZATION OF AND SAPONIN PRESENT a- IN Two proteins with a-amylase inhibitors, A-I and B-2, were partially purified from Colocasia esculenta tubers (extraction with 1% PVP, 80 per cent ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sephacel and chromatography on Sephadex G-IOO). Using gel chromatography on G-I00, the molecular weight of A-I and B-2 were found to be approximately 17 000 and 19 000 respectively. The inhibitors inactivated Cl-amylases of animal origin, but had no effect on fungal amylase. Inhibitor A-I also exhibited activity towards plant amylases, while inhibitor B-2 evidenced no activity on plant amylases. Inhibitor A-I was most active (with human-salivary amylase) at pH 6. Inhibitor A-I was completely destroyed at temperatures above 50? C, while inhibitor B-2 was stable up to 70? C. A steroidal saponin was also isolated from the tubers of Colocasia esculenta. The structure was elucidated mainly with TLe, IR. (Infrared spectroscopy) and GC-MS (Gas Chromatography - Mass Spectroscopy) spectroscopic analysis. The compound was determined to be gamma-sitosterol. lI8 CHAPTER Bl-l: a-AMYLASE INIDBITOR (AI) LITERATURE REVIEW B1.1.1 Introduction Many edible plant species consist of compounds that inhlbit enzymes, principally hydrolases. Most of these inhibitors are proteins by nature, which particularly inhibit enzymes by forming complexes that block the active site or modify enzyme conformation, ultimately reducing the catalytic function. In the previous section (Section A), a-amylase inhibitor activity was identified as one of the anti-nutrients present in Amadumbe. In this section, a-amylase inhibitors in Colocasia esculenta will be isolated from white Esikhawini tubers and partially characterized. B1.1.2 a-Amylase inhibitor Starch is a storage carbohydrate present in seeds and tobers of numerous plants (Teotia et al. 200I). Starch consists of two components: amylose and amylopectin. Amylose is a linear glucose polymer, which contains a-l,4 linkages and is a non branched polymer. Amylopectin, on the other hand, is a highly branched polymer of glucose in which linear chains of a-l,4 glucose residues are interlinked by a-l,6Iinkages [Buleon et aI., 1998]. Prior to starches being absorbed, they frrst need to be broken down into glucose and smaller oligosaccharides. Digestive enzymes (amylase and isomaltase) are responsible for this catalytic reaction (MarshaIl and Lauda, 1975; Choudbury et al., 1996). Amylases (a-I,4-glucan-4-glucanohydrolases, EC 3.2.1.1) are a group of glycoside hydrolases widely distributed in microorganisms, plants and animal tissues (Whitaker, 1988; Payan, 2004). a-Amylases hydrolyze long, complex, starch chain molecules to release maltotriose and maltose from amylose and maltose, glucose and "limit dextrin" from amylopectin (Moore et aI., 2005). Without calcium, the a-amylases which are 1I9 calcium metalloenzymes, are completely unable to function. MacGregor et al., (2001) commented that these amylases are crucial to the carbohydrate metabolism of many autotrophic and heterotrophic organisms. a-Amylase catalyzes the break down of 0.-(1,4) glycosidic linkages found in starch components and other related carbohydrates (DoleekoVli-MareSova et al., 2005). In autotrophic organisms, sugars are gradually released from starch which has previously been stored, thus providing energy for proper growth. Amylases have established many valuable functions within society, some of which are disease testing, fruit ripening and malt production (Teotia et al., 2001). a-Amylases also play a major part in breaking down starch in germinating seeds (Jones and Jacobsen, 1991). Usually, the major constituent of man's diet is carbohydrate and the main carbohydrate ingested is starch. a Amylases are the most important means heterotrophic organisms use to digest starch (Silva et at., 2000). Several compounds found in nature are responsible for inhibiting enzymes, especially the hydrolases (Kokiladevi, 2005). Current investigations in the field of proteinase and amylase inhibitors and the growing interest in these phytochemicals in industry and pharmacy have led researchers to inquire directly into their exact biological function. Some of these inhibitors are proteins which decrease specific enzyme activities, such as the inhIbitors ofproteases and amylases (Whitaker and Feeney, 1973). In 1934, Chrzaszcz and Janicki identified AI in wheat and since then, numerous studies have recognized the fact that a-amylase inhibitors occur naturally in a wide variety of plants. Richardson (1991) and Franco et al. (2002) proposed that a-Amylase inhibitors may simply be classified by their tertiary structure into six different classes, which researchers identify as lectin-like, knottin-like, cereal-type, Kunitz-like, y-purothionin like and thautamin-like. The molecular weight, disulfide bond content, three-dimensional structure and stability to heat and denaturing agent (Teles et at., 2004) differentiate among these families. Each family of a-amylase inhIbitors shows particular specificity features: 120 B1.1.2.1 Lectin-like a-amylase inhibitor (AI) Lectin-like a-amylase inhibitors have been purified and characterized from different varieties of common bean (Phaseolus vulgaris) [Le Berre-Anton et aI., 1997; Lee et al., 2002]. These inhibitors have two variants with a high degree of sequence homology (Suzuki et aI., 1994). Three inhibitors are encoded by two different alleles. One of these inhibitors inhibits both mammalian and insect-amylases, whereas another inhibits different insect a-amylases (Franco et al., 2002). Bl.1.2.2 Knottin-Iike a-amylase inhibitor The knottin-like family contains the AI from the Mexican crop plant, amaranth (Amaranthus hypochondriacus). This AI is the smallest known, natural, proteinaceous a amylase inhibitor (Chagolla-Lopez et al., 1994). The seeds of the amaranth inhibit insect a-amylases, but are inactive against mammalian enzymes (pereira et aI., 1999). The tertiary protein structure shows knots particularly rich in disulphide bridges. This structure can exist both as individual miniproteins of around 32 amino-acid residues and as domains in larger molecules (Svensson et at., 2004). Bl.1.2.3 Cereal-type a-amylase inhibitor This is a large protein family which includes a-amylase inhibitors from cereal seeds (Barber et al., 1986; Garcia-Maroto et aI., 1991). Members of this family are known for their activity on a-amylases from mammals and insects, but have also shown inhibitory activity against a-amylases from birds and bacteria (Franco et al., 2002). 121 Bl.1.2.4 Kunitz-Iike a-amylase inhibitor Macedo et al (2007) defined plant Kunitz inhibitors as proteins (Mr - 18,000 - 24 000 Da) which have one or two polypeptide chains. They have a low cysteine content, generally with four cysteine residnes organized into two disulphide bridges. Bl.1.2.5 1-Thionin-Iike a-amylase inhibitor The major utilization of the y-thionine a-amylase inhibitor family is in processes promoting plant-defense through several means: adaptation of membrane permeability (Castro et al., 1996), blockage of protein synthesis (Mendez et al., 1990) and digestive enzyme inhibition (Wijaya et al., 2000). y-Purothionin-like proteins have shown specificity to inhibit insect digestive a-amylase (Bloch and Richardson, 1991). Bl.l.2.6 Thaumatin-Iike a-amylase inhibitor Proteins from the thaurnatin-like a-amylase inhibitor family have the ability to modify the properties of fungal cell walls. Schimoler-O'Rourke et aI., (2001) identified Zeamatin as a 22 kDa protein isolated from lea mays, pointing out that it has noteworthy amino acid homology to taumatin 3-like proteins. It inhibits insects, but not mammalian u amylases (Svensson et al., 2004). Amylase inhibitors prevent dietary starches from being absorbed and are therefore also known as starch blockers. Reports on purified AI have shown that intraluminal amylase activity is significantly inhibited when perfnsed into the duodenum (Layer et at., 1985). When dietary starch is ingested with AI, it reduces the post-prandial increase in glucose significantly in both normal and diabetic patients (Layer et at., 1986). Diarrhea may result from undigested starch in the colon, which is the cause ofhigh amounts of amylase inhibitors (Boivin et at., 1988). 122 a.-Amylase inhibitors could function as part of the defense mechanism in plants. In leguminous plants (Giri and Kachole, 1998; Melo et al., 1999) and cereals (Franco et al., 2000; Yamagata et al., 1998), the role of amylase inlnbitors as plant-defense proteins is pronounced and has received much focus (Gatehouse et al., 1986; Farmer and Ryan, 1990). a-Amylases and proteinases are inactivated by these inlnbitors in the insect gut, thereby acting as insect anti-feedants. This interaction is believed to make plants less palatable. Indeed, it can even be lethal to insects. Thus, these inlnbitors present the plants with some selective advantages. Increasing natural defense mechanisms in plants can enhance agriculturaI activity and food safety by decreasing intensive use of pesticides (Huang et al., 1997; Chen et al., 1999). These inlnbitors, however, often show confined specificities: a given inhibitor may inhibit the major digestive enzymes of one insect species, but not of another (Morton et al., 2000). This specificity has been widely investigated, with some inhibitors capable of acting against insect a-amylases or against mammalian enzymes only (Franco et aI., 2000). As a resuh, a-amylase inhibitors show potential for utilization in several fields, including protection of crops, obesity and treatment of diabetes (Tormo et aI., 2006). Diabetes mellitus is a metabolic disorder of multiple aetiology characterised by chronic hyperglycaemia with disturbance of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both (WHO, 1999). Glucose peaks occur after the intake of a meal in animals. a-Amylase inhibitors can reduce these peaks until the body is capable of processing the glucose. This is achieved by reducing the speed with which a-amylase can convert starch to simple sugars. Breuer (2003) held that this is especially important for diabetics who exhibit low insulin levels which hamper the prompt removal of extracellular glucose from the blood. Ali et al (2006) observed that reducing hyperglycaemia, using extracts of six selected Malaysian plants, after meals is one therapeutic approach which could be adopted in the treatement ofdiabetes. Clinical use of inhibitors of intraluminal a-amylase activity has appeal because, in theory, controlled reduction of starch digestion could influence carbohydrate uptake in 123 diabetes mellitus or obesity. Octivio and Rigden (2000) referred to a-amylase and its inhibitors as 'drug-design targets' from which compounds for the treatment of diabetes, obesity and hyperlipaemia could be developed. Btl.3 Aim and outline of Section B-1 Amylase inlnbitors have been extracted from several types of plants, especially those in the cereal and legume family. In contrast to a-amylase inhibitors in cereal (Roy and Gupta, 2000; Heidari et al., 2005; Muralikrishna and Nirma1a, 2005) and legumes (Giri and Kachole, 1998; Melo et ai, 1999), which have been extensively studied, little is known about the structural features and properties of tuber a-amylase inhibitors. The aim of Section B-1 was therefore to extract, isolate and characterize a-amylase inhibitors from Amadumbe. The objectives were: ? the extraction, isolation and partial purification of a-amylase inhibitors through ion-exchange and gel chromatography; ? to undertake kinetic studies (pH and temperature optima); and ? to investigate the selectivity of inhibitor action on different a-amylases. 124 CHAPTER BI-2 MATERIALS AND METHODS B1.2.1 Introduction This chapter gives a brief description of the materials and methods used to isolate, partially purify and characterize a-amylase inhibitors from white Colocasia esculenta grown in Esikhawini, Zululand, South Africa. (See Appendices A and B for details). B1.2.2 Materials The white variety of Amadumbe (Colocasia esculenta) tubers was obtained from the local maIket in Esikhawini, Kwazulu-Nata1, South-Africa. Commercially available amylases (human saliva, type IX-A; porcine pancreatic, type I-A; sweet potato, barley, Bacillus species, type IT-A; Aspergillus oryzae) and all other reagents used were obtained from Sigma Chemical Company. B1.23 Methods (See Appendix B for details ofmethodology.) 125 B1.2.3.1 Extraction and purification of a.-amylase inhibitor The scheme for the extraction and purification is shown in Figure B-1. Plant material IDefatted with hexane 11 %PVP(2x) ICrude extract 1Ammonium sulphate precipitation Extract 1 Ion-exchange chromatography 1 Gel chromatography Figure Bl-2.1: Extraction protocol for isolating a.-amykase inhibitor from Amadumbe tubers Tubers with no physical signs of infection were washed, peeled, cut into small pieces (2 cm x 3 cm) and dried at 40? C for 24 hours. The dried material was milled (68 mesh) and the flour defatted with hexane and air-dried. Twenty grams of the defatted flour were added to 100 ml of distilled water (containing one per cent PVP), stirred for two hours and filtered. The residue was re-extracted and the combined filtrate centrifuged at 12000 g for 20 minutes. 126 B1.2.3.1.1 The supernatant (designated as the erode extract) was subjected to 80 per cent CNThhS04 saturation and left overnight at 4? C. Protein pellets obtained after centrifugation (12 000 g x 20 minutes) were redissolved in a minimum volume of phosphate buffer (0.02 M, pH6.9, containing 0.3 M NaCI), dialysed extensively against the buffer (designated the ammonium sulphate extract) and analysed for amylase inhibitor (AI) activity. Ion-exchange chromatography The ammonium sulphate extract was further pu,ified through ion-exchaoge chromatography (6 cm x I.I cm DEAE-Sephacel, equilibrated with 0.02 M phosphate buffer). The column was eluted with a linear NaCl gradient of 0-0.5 M at the flow rate of 20 mIIh and 5 mI fractions were collected). The absorbance of the effluent was monitored at 280 nm. Individual peaks were pooled and analysed for protein and AI activity. B1.2.3.1.2 Gel chromatography Two peaks (A and B, Figure B1-3.2) with AI actlVlty were then separately chromatographed on a Sephadex G-100 column (35 cm x 1.1 cm, equilibrated with the phosphate buffer and eluted with the same buffer at a flow rate of 15 mllhr. Five mI fractions were collected and the absorbance at 280 run was determined). Pooled fractions were analysed for protein and AI activity. Fractions (A-1 and B-2, Figure B-4) with AI activities were collected, dialysed extensively, freeze-dried and dissolved in de-ionized water. B1.2.3.2 Enzymefmhibitor assay The method described by Bernfeld (1955) was used to assay for a-amylase and AI activities. One unit of amylase is defined as the amount of enzyme that will liberate 1 !lmol of maltose from starch under the assay conditions (pH 6.9, 37" C, 5 min). Inhibitory activity is expressed as the percentage of inhIbited enzyme activity out of the total enzyme 127 B1.2.3.3 activity used in the assay. The incubation time of the enzymes with the inhibitor fractions was 20 minutes at 37? C. Molecular weight determination Proteins were determined using the Sigma kit. Molecular weight was determined by gel chromatography on Sephadex G-lOO, under the conditions described by Sigma. Cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), alcohol dehydrogenase (150 kDa), and ~-amylase(200 kDa) were used as molecular weight markers. Bl.2.3.4 Kinetic studies B1.2.3.4.1 a-Amylase inhibitor specificity a-Amylase inhibitory activities were estimated as in Section B.1.2.3.2, using a-amylases from human saliva, sweet potato, barley, porcine pancreas, Bacillus and Aspergillus oryzae. The AI with the corresponding amylase was pre-incubated for 30 minutes at 37? C in phosphate buffer pH 6.9 before adding starch to initiate the reaction. B1.2.3.4.2 Optimum pH The pH optimum of the inhibitor was determined by varying the pH of the test reaction mixture using the following buffers (0.1 M): sodium acetate (pH 5), sodium phosphate (pH 6-7), Tris-HC! (pH 8) and glycine-NaOH buffer (pH 9-10). To determine the pH of the (I-amylase inhibitor, it was pre-incubated in different buffers (pH 1-10) for five minutes. B1.2.3.4.3 Optimnm temperature The temperature optimum of the iubibitor was evaluated by measuring (I-amylase activity at different temperatures (20 - 1000 C) in phosphate buffer (pH 6.9). 128 BI.3.1 BI.3.2.1 CHAPTER BI-3 RESULTS Introduction In this chapter, the results for the isolation, partial purification and characterization of the two a-amylase inhibitors from Amadumbe are presented. Extraction and purification of a-Amylase inhibitors The overall results of the purification procedure of a-amylase inlnbitors from Colocasia esculenta have been summarized in Table BI-3.1. Preliminary studies (Figure B-1) suggested that 80 per cent ammonium-sulphate saturation was best for salting out the inhibitor protein from the crude extract. Table BI-3.1: Purification of amylase inhibitors from Amadumbe Crude extract Ammonium sulphate DEAE sephacel: 20.9 18.5 62 56 2.9 3.02 100 88.5 1.04 Peak A PeakB Sephadex G-lOO Peak Al PeakBl 7.2 5.9 3.7 5.6 54 50.5 41.8 39.8 7.5 8.56 11.29 12.83 33.44 28.22 17.70 14.83 2.59 2.93 3.89 4.42 Note: The inhibitor units were calculated using human-salivary amylase. 129 '00 1 so 80 C 70 :~? ,'-" 60=ts ~~]] 50 ._ f ..l:"- .5~ ";;~ 40 ~'I- >!i. 300 20 'A 0 0 20 40 60 80 '00 120 % Ammonium su1fate saturation Figure Bl-3.1: Ammonium sulphate precipitation of the Amadumbe crude extract Key inhibitor activity (.) total protein ( ? ) 130 The profile of the ion-exchange chromatography is shown in Figure BI-3.2. Only two (A and B) of the five protein peaks showed a-amylase inlnbitory activity. ,. 0 0.5M 8 NaCI gradient , EE ' Cc ,. lil N @2, .. u ,.c .. .a ::; 1. - .. ' .a et ,. o . , 0 , " , '"0 No of tubes Figure BI-3.2: Ion-exchange chromatography of extract on DEAE-Sephacel 131 The inlnbitor peaks (Figure BI-3.3) from the DEAE-Sephacel chromatography were subsequently separated by gel filtration. Only two (AI and B2) of these proteins had inhibitory activity against human salivary a-amylase. Gel filtration Sample A 0"'1 3I E oo07l ~o"'l @0"1 4Jo~ 2 S0.03 ~ ... i< 0.02 ~ I 0.01 \ "/ \;\...00 5 10 15 '" 25 >l No of tubes Gel filtration Sample B 0.'" 2 0.07 ~ 0.06 o '"N 0,05 ~ 0.04 C ..i! 003 o co ~ 002 001 1 3 4 302515105 o+----......__l---,~_-~--~-+___I_,......l_ _4_.....--~ o No of tubes Figure BI-3.3: Sephadex G-lOO column chromatography offraction A and B after ion-exchange chromatography on DEAE-Sephacel 132 The molecular weight of AI-AI and AI-B as determined by gel filtration on Sephadex G 100, was estimated to be about 17 kDa and 19 kDarespectively. 1000 -.. {g100 :le: --- - ..c: .21 ~ .... IIIG 10 ~ o ::2 1 I I I I I. - I I -- V- I I?L--- iN II i I I I I I I I I I I I iI I o 1 2 VeNa 3 4 5 Figure Bl-3.4: Standard Log Molecular Weight graph using gel filtration Standards cytochrome C (12 400) alcohol dehydrogenase (150 000) carbonic anhydrase (29 000) J3-amylase (200 000) 133 B1.3.2.2 Kinetic studies The inhibitory activity of the purified proteins on amylases from different sources is presented in Table BI-3.2. The two proteins were found to inhibit mammalian and bacterial amylases. While protein Al inhibited amylase from plant sources, protein B2 showed no such effect. The two proteins could not inhibit fungal amylase. Table Bl-3.2: Effect of a-amylase inhibitors present in Amadumbe against amylases from different sources Source of amylases Percentage inhibition Al B2 Human salivary 62 56 Barley 56 0 Sweet potato 4.7 0 Bacillus species 10.2 23 Aspergillus species 0 0 Porcine pancreas 28.5 48.5 134 The effect of temperature on the two proteins is presented in Figure B1-3.5. The two proteins were most active at 40? C. They were completely inactivated at temperatures above 80? C. 30 25 20 ? \~~ 15"" 10 \ \ 5 0+-----4~-_-----_-_4--_...:::::=__-_-__--_ 30" <0" 70" TempendUre I"C) SO" 100 " Figure BI-3.5: Effect of temperature on the activity of Colocasia esculenta a amylase inhibitors. Key AI-AI (+) 135 Optimum pH for amylase inlnbitor activity in Colocasia esculenta is shown in Figure Bl-3.6. A two-peakpattem was found, which showed an optimum at pH 4.0 and 6.0. 35 3D 2S 120 ..,. ,. ,. ? ? ? 2 4 ? ? ,. 12 pH Figure BI-3.6: Effect of pH on the stability of Amadumbe a-amylase inhibitor The enzyme solutions at varions pH values were incubated at 25? C for 10 minutes and residual activity was measured as descnbed in Appendix B. 136 Bl.4.1 B1.4.2 CHAPTER B1-4 DISCUSSION Introduction In this chapter, the results of the isolation, partial purification and characterization of the two a-amylase inhtbitors from Amadumbe are discussed. Isolation of a-amylase inhibitor A 3.89- and 4.42-fold purification of AI-AI and AI-B2 respectively was obtained. The proteins had specific activity of I 1.29 and 12.83 respectively. The purified a-amylase inhtbitors (AI-AI and AI-B2) from Colocasia esculenta showed a molecular weight of approximately 17 kDa and 19 kDa respectively. The little available information on a-amylase inhibitors present in Colocasia antiquorum (Sharma and Pattabiraman, 1980), taro (Seltzer and Strumeyer, 1990) and sweet potato (Rekha et al., 2004) seemed to indicate that most tubers had two proteins that showed inhibiting activity. The molecular weight of these proteins ranged between I I and 25 kDa. B1.4.3 Kinetic studies B1.4.3.1 Action of inhibitors on different a-amylases a-Amylase inhibitors show strict target enzyme specificity and recognize ouly one out of several closely related isoenzymes or enzymes from different species (Weselake et aI., 1983; Franco et aI., 2000). Payan (2004) observed that amylase-inhtbitor complexes have general features of inhtbition on different amylases: (i) the inhibitor inhibits primarily via interactions with the enzyme substrate active site (ii) side-chains originating from the 137 B1.4.3.2 inhibitor molecule generally occupy the subsites of the enzyme (iii) structmal elements involved in the inlnbition action are likely to correspond to flexible components of the free structures of the molecules. Literature (Sharma and Pattabiraman, 1980, 1982; Ida et al., 1994) indicates that many AI present in tubers are active against mammalian amylases, but exlnbit no activity on plant amylases. It is apparent that the two AI present in Amadumbe will complement each other, providing the Amadumbe tubers with a wider spectrum against intruders. It is apparent that both inhIbitors had no effect on fungal amylase. Amadumbe grow in moist, humid conditions and fungal infections are prevalent. It is possible that fungus has become resistant to the action of a-amylase inhibitors. Sharma and Pattabiraman (1982) have reported simiIar results for Dioscorea alala. Bifunctional properties have been demonstrated by a number of inhIbitors and have therefore received particular attention as appealing candidates for pest-contro!. This biological activitiy of inhibitors, inhIbiting both serine proteinases and a-amylases, is very useful (Maskos et al., 1996). a-Amylase inhibitors show great variety in their effectiveness against target enzymes. a-Amylase inhibitors found in wheat (Franco et aI., 2000), barley (Richardsoll, 1991) and Indian finger millet (Campos and Richardson, 1983) efficiently inhibit a-amylases from different insect sources. Thus, these a-amylase inhibitors can play a key role in plant defense against pests and pathogens. Effect of temperature on inhibitor activity Temperature is one of the most important parameters that affect the rate of enzyme hydrolysis. The optimum temperature displayed for both inhIbitors was 40? C and, at extreme temperature, inhibitors became inactive. Optimum temperature for the navy-bean amylase inhibitor was 37? C, but activity was lost at 90? C (Hoover and Sosulski, 1984). A similar optimum temperature of 37? C was also observed for the a-amylase inhibitor from a Pacchyrhizus erosus tuber (Noman et al., 2006). It can be concluded from these 138 Bl.4.3.3 results that amylase inhibitors are fairly heat-stable (PratInbha et al., 1995) and that the residual activity in processed Amadumbe (Section A) could be attrIbuted to this property. Effect ofpH on the interaction of amylase with the inhibitor The AI protein was not active at acidic pH. The proteins isolated from Colocasia antiquorum (Sharma et aI., 2006) were basic proteins. Because of the two peaks of optimal pH points, it is possible that isoenzymes are present. A similar optimum pH 7.0 was observed for the a-amylase inhibitor from the P. erosus tuber (Noman et al., 2006). The partially purified and characterized a-amylase inhibitors from Amadumbe showed similar properties when compared with a-amylase inhibitors from other tubers. Although the Amadumbe a-amylase inhibitors showed activity against mammalian amylases, processing greatly inactivated the biological activity of the inhibitors. 139 B2.1.1 B2.1.2.1 CHAPTER B2-1: SAPONIN LITERATURE REVIEW Introduction Many different secondary metabolites are synthesized collectively by plants. This can either be as a response to pathogen attacks and stress or part of the plant's normal growth. Although these secondary metabolites are not required for growth and reproduction, they are important in that they offer the plant selective advantages: for example, restraining the growth of neighboring plants or protecting the plant against pests, pathogens and stress (Wink, 1999; Morrissey and Osbourn, 1999). Saponin - chemistry Saponins are a pharmacodynamic group of secondary metabolites with a wide spectrum of biological activities. They are found in more than 90 plant families (Sondhia, 2005), such as peanuts, lentils, lupins, alfalfa, oats and spinach (Fenwick and Oakenfull, 1983; Huhman and Sunmer, 2002; Woldemichael et aI., 2003). Saponins are characterized by surfactant properties because they contain both hydrophobic and hydrophilic components and, in most cases, give stable, soap-like foams in aqueous solutions. Saponins are glycosidic compounds containing a carbohydrate and a non-carbohydrate unit in the same molecule. An acetal linkage joins the carbohydrate residue to a non-earbohydrate residue or aglycone at carbon atom position 1. The sugar component is called the glycone. Saponins are surface-active compounds because of a lipid-soluble aglycone and water-soluble sugar chain(s) in their structure. This aruphiphilic characteristic gives saponins detergent, wetting, emulsifying and foaming properties (Ibanoglu and lbanoglu, 2000; Sarnthein-Graf and La Mesa, 2004; Wang et aI., 2005) 140 The chemical structure ofits sapogenin (aglycone) determines the type to which the saponin belongs - steroidal or tritezpenoid (Figure B2-1.1). Saponins are glycosides because they contain one or more sugar chains attached to the aglycone backbone (Hostettmann and Marston, 1995). Saponin Glycone Glycoside (sugar) Aglycone Sapogenin Neutral saponin l Steroids Acid saponin l Triterpenoids Figure B2-1.1: Types of saponin (Friedli, n.d) Both types of sapogenins are synthesized from a similar pathway, involving the head-to tail coupling of acetate units. However, after the formation of the triterpenoid hydrocarbon, squalene (Holstein and Hohl, 2004), there is a split in the pathway that leads to steroids in one direction and to cyclic triterpenes in the other (Figure B2-1.2) 141 acetoacetyl CoA spiroketal steroid acetylCoA + (2)NADPH HMG-CoA ' mevalonate HMlrCoA reductase 1(3) ATP Co, isopentenyl pyrophosphate 1 geranyl pyrophosphate 1 squalene 1l2.1.2.2 pentacyclic triterpenoid Figure 82-1.2: Pathway to biosynthesis oftriterpenes (adapted from Focosi, 2005) Triterpenoid saponin Triterpenoid are a large group of compounds with 30 carbon atoms with four basic ring skeletal structures. The large group of compounds to which triterpenes belong are arranged in a four- or five-ring, planar-base molecule, containing 30 carbons with several oxygen attached. It is believed that squalene is the prescursor ofall terpenoid compounds including triterpenoids. Initial steps in the synthesis of triterpenoid saponins in plants include the cyclization of 2,3-oxidosqualene via the isoprenoid pathway, giving rise to a number of different potential products (Hostettmann and Marston, 1995; Haralampidis et aI., 2002). The skeleton formed is then determined by the type of cyclase that is involved 142 in the cyclization reaction. There are about 20 groups of triterpenes, the specific structure of the triterpene determining to which group it belongs. HO '""&J... .".( , 't1lCllD HO et 4."~ C-.w> HO Figure B2-1.3: Basic aglycone (sapogenin) skeletons -triterpene (Oleszek and Bialy, 2006) 143 12.1.2.3 Steroidal saponin The triterpenes and the plant steroids are broadly descnbed as saponins (Kelly, 2005). Sterols fonn an important group among the steroidal saponins. Like triterpenes, phytosterols are synthesized in the isoprenoid pathway. Triteq>enes and plant sterols are differentiated by the strncture of the carbon skeleton: triterpene saponins have a 30 carbon skeleton and sterols have a 27/29-earbon skeleton (Ukpabi and Ukpabi, 2003). Sterols were initially descnbed as compounds having a structure similar to that of cholesterol, but this definition did not take their stereochemistry into account. Nes (1977) proposed a new definition, descnbing sterols as compounds developing from squalene or its oxide by a cyclization process. Therefore, the biosynthetic route to plant sterols also follows an isoprenoid biosynthetic pathway with isopentenyl pyrophosphate, derived from mevalonate, as the key building block (Piironen, 2000). However, in photosynthetic organisms (as opposed to yeast and fungi), it differs in that the important intermediate in the route from squalene is cycloartenol rather than lanosterol (GIbbons et at., 1971). The basic sterol from which other sterol structures are defined is 5a-cholestan-31J-01 (Sterols, 2008). The phytosterols include campesterol, beta-sitosterol, and stigmasterol etc. Steroidal saponins are comprised of two major classes: furostanol glycosides with an open side chain at C-22 and spirostanol glycosides with a closed spiroketal ring at C-22 (Figure B2-1.4). Furostanol glycosides are the foundation from which spirostanol glycosides are fonned through hydrolysis of the C-26 sugar and spontaneous cyclisation of the side chain from the spiroketal (Inoue and Ebizuka, 1996). 144 ? 82.1.2.4 or (PH sn .. (pa'MsI) Figure B2-1.4: Basic aglycone (sapogenin) skeletons - steroidal (Oleszek and Baily, 2006) Biological activities Becanse they have a number of both positive and negative properties, saponins have attracted a great deal of interest (Shi et al., 2004). These properties include sweetness and bitterness (Kitagawa, 2002; Heng et a!., 2006), foaming and emulsifying characteristics (Price et aI., 1987), anti-nutritional effects (Fenwick et aI., 1992), pharmacological and medicinal properties (Attele et a!., 1999) as well as haemolytic properties (Oda et a!. 2000; Sparg et al., 2004). One of the most researched attributes of saponins is their ability to swell and rupture erythrocytes, causing a release of the pigment haemoglobin 145 (the in vitro haemolytic activity) (Oda et aI., 2000). Although the concept that they are harmful to human health has been questioned (Reddy and Pierson, 1994), saponins have been reported to retard growth in animals (Cheek:e 1971; 1976). The ability of saponins to lower cholesterol, demonstrated in animal (Matsuura, 2001) and human trials (Iones et aI., 1997) has been extensively researched. This trait of saponins has been attributed to their inhibiting cholestrol absorption from the small intestine or their inhibiting reabsorption of bile acids (Oakenfull and Sidhu, 1990). Clinical studies have suggested that the health-promoting components of saponins affect the immune system, impacting this system through adjuvant activity (Cheeke, 1999). The ability of saponins to operate as immunological adjuvants by improving the immune response to antigens has been recognized since the 1940s (Bomford et al., 1992; Francis et al., 2002). It is through the immunde response that the immune system helps to protect the human body against cancers. A number of triterpene and steroid saponins have shown anti-eancer activity (Berhow et al., 2000; Kerwin, 2004). A saponin-rich diet offers several benefits, including a reduction in the occurrence of renal stones, the inhibition of dental carries, improved platelet aggregation and a positive response to treating hypercalciuria in humans (Shi et al., 2004). 2.1.3 Aim and outline of Section B-2 Saponins can be found in a wide variety of plants including alfalfa (Cheeke et aI., 1977), soybeans (Berhow et al., 2002; Kitagawa et al., 1998) and legume seeds (Price et al., 2006). Little is known about levels of saponins in Colocasia esculenJa (Amadurnbe). The aim of Section B-2 was to extract, isolate and identify saponins from Amadurnbe. The objectives were: ? to extract and isolate saponin using chromatographic methods (TLC and column chromatography); ? to elucidate saponin structure, using spectroscopy (IR and GC-MS). 146 B2.2.1 B2.2.2 B2.2.3 CHAPTER B2-2 MATERIALS AND METHODS Introduction This chapter gives a brief description of the materials and methods used to isolate, partially purify and characterize a saponin from white Colocasia esculenta grown in Esikhawini, Zululand, South Africa. (See Appendix B for methodology). Plant material White tubers from Colocasia esculenta (Amadmnbe) were collected from the local market at Esikhawini, Kwazulu-Natal, South Africa. The plant materials were collected between January and March 2006. Methods (See Appendix B for details of methodology) 147 B2.2.3.1 Saponin extraction and isolation The extraction procedure is illustrated below. Plant material 1 I kg air-dried plant material was extracted with 95 per cent ethanol (repeated five times) 1 followed by filtration vacuum evaporation (40? C) Crude extract I Fractionate / n-Butanol extract (saponin extract) TLC and column chromatography analysis I ~ I Chloroform extract 1 TLC analysis Figure B2-2.1: Extraction protocol for obtaining native saponins from Amadumbe tubers 148 82.2.3.2 82.2.3.2.1 B2.2.3.2.2 Air-dried and powdered Amadumbe tubers were extracted with 95 per cent ethanol. The ethanolic extract was successively partitioned with chloroform and n-butanol respectively. The n-butanol-saponin fraction was then analysed by TLC. TLC procedure was optimized using the eluents chlomform:methanol:water in varying ratios. It was observed that there were many compounds present in the fraction. although at different Rt values. The fraction obtained when the mobile phase composition of chlomform:methanol:water was 65:5:10 and 85:10:5 gave the best TLC separation. The TLC plates were evaluated and the Rf values determined. Only one of the compounds was reproducible by comparison with the standard Rt values of 0.64. Similar fractions from the TLC plates were combined by scraping off and were analysed by spectroscopic methods for identification. Chromatographic methods Thin-layer chromatography TLC was performed on Silica gel 60 F254 aluminium baked sheets of 20 cm by 20 cm (Merck). Detection was undertaken. using iodine crystals and anisaldehyde/sulphuric acid! acetic acid (90:5:S v/v). Column chromatography The n-butanol fraction was subjected to column chromatography, using Silica gel 70-240 mesh. Chloroform (CHCh) was initially used as the eluting solvent. Polarity of the solvent was increased by adding methanol until a ratio of 50:50 was reached. Fractions were pooled according to TLC analysis. 149 B2.2.3.3 Spectroscopy B2.2.3.3.1 Infrared spectroscopy (IR) IR is a technique using the interaction of substances with infrared electromagnetic radiation to identify different functional groups in an unknown substance. IR spectra were measured using a PeIkinElmer Spectrum 100 series and attenuated total reflectance (ATR) as the sampling technique. Liquid isolates were used. 82.2.3.3.2 B2.2.3.3.3 High-Pressure Liquid Chromatography with UV detector analysis (HPLC-UV) Reversed-phase HPLC analysis, usmg a Sbimadzu liquid chromatograph with a Teknokroma nucleosil 100 Cl8 column (5Jllll x 25 mm x 4 mm) was used to detect conjugation in the isolate. Detection was conducted by means of a Prominence Diode Array detector, at room temperature. The mobile phase used for HPLC experiments was 10 per cent acetonitrile (CH3CN) and water. The samples were injected, using a Prominence autosampler, and were monitored for 15 minutes, at flow rate of 1.0 mlImin. Gas chromatography mass spectrometry (GC-MS) GC-MS is an analytical technique used to separate the ions according to mass/charge (m/z). Ionization of the molecules by high energy electrons in an electric field is used to determine molecular weight. GC-MS was run on an Agilent 6890 GC system, including an HP 5973 MS instrument, using a J & W lIP5-MS fused silica capillary column (30 m x 0.25 mm x 0.25 /lID) under the following conditions: ? filament current: 4.2 A:, ? column temperature: 1801260? C; ? programmed increase: 5? C/min; ? carrier gas: He; ? head pressure: 12 psi; ? El-MS: 70 eV; 150 82.2.3.3.4 ? ion source temperature: 250" C. Silica gel (200-300 mesh and 10-40 p.m). RP-IS (40-63 p.m) and Sephadex LH-20 were used for column chromatography. Nuclear magnetic resonance (NMR) NMR is a method which has beeo developed to determine molecular structure by absorption of radio waves in the presence of a strong magnetic field. NMR spectra were run on a Broker AM-4oo (for IH and 13C_NMR) instrument, with tetramethylsilane (TMS) as internal standard. 151 B2.3.t B2.3.2 CHAPfER B2-J RESULTS Introduction In this chapter, the results for the isolation, partial purification and characterization of saponin BI from Amadumbe are presented. Extraction and purification of saponin The crude ethanolic extract of Amadumbe was partitioned between chloroform and n butanol. The n-butanol fraction was analysed by TLC. Figure B2-3.1 shows the separated spots. 152 /) 0.93\~ 0 0 0.80"C =:= Clg, a Cl 0 0,64 o Bl 0.64.. BlS "C ... =i;. <= .. -... .. =<= -'".-l:l Baseline Extract Extract Figure B2-3.1: Thin-layer chromatographic examination of saponin compounds from an Amadumbe (Colocasia esculento) tuber Aliquots of 10 j.il from crude extract were cbromatographed on a gel 60 Fm plate in the solvent system CHCl):MeOH:H20 [65:35:10]. The spots were located by means of the standard references. Comparison of Rf value makes it possible to research complex mixtures qualitatively (Fernand 2003). The compounds with an Rf va1ue of 0.64 (saponin B1) were scraped offand dispersed in chloroform. The combined saponin Bl was identified using IR and GC-MS. The spectra were compared with the spectra available in the horary of the computerized GC-MS. 153 10 .66 .6S 380.0600 , .OS 100012001400160018002000 - 1111 3200 1465.25 "'"SS 99.9 98 96 94 92 90 Si .. ... %T 82 sa 71l 76 74 72 70 67.9 _.. 3600 ~1 Figure 82-3.2: Infrared spectrum of saponin HI fraction of white Colocasia esculenta (Measurements were carried out at 27? C) The IR spectrum showed a peak at 3324 cm-I (OH), 2940 (C-H), an ether linkage 1000 1100 cm-I, 2873.65 - 2932.19 cm-I (C=H stretching) and 1465.25 (C=C stretching). This spectrum suggests that there are functional groups present in the compound saponin Bl. The GC-MS of saponin BI (Figure B2-3.3) showed a molecular-ion peak with a retention time of22.54, which indicates the molecular formula C2<;I!5QO. 154 Figure 82-3.3: Chromatogram ofsaponin BI from Amadumbe anaylsed by GC-MS GC-MS was used to identify the isolated compound. The GC-MS spectrum showed a molecular-ion peak at 414 mJz, indicating a molecular weight of 414 dalton. 155 AIuldInclI J 50 100 414 S 2lIll 2150 3110 3lIll ... 4liO 500 5IiO #109486: .gamma-SiIDsIerDI 41" 0 ! ~ 50 150 . 2lIO 2150 3110 3lIll ... 4liO 500 -AIuldInclI #10380: .beta.-8iIoSleroII G 414 81 101 0 1IlIz- 50 100 150 2lIll 2150 450 500 5IiO Figure B2-3.4: Gas Chromatographic profile of saponin Dl analysed by GC-MS Identification of components was confinned by comparison of collected mass spectra with those of authenticated standards and spectra from the National Institute for Standards and Technology (NIST) mass spectral library, Search Version 2.0. The GC-MS spectra obtained for the isolated compound gives two suggestions as gamma-sitosterol and beta-sitosteroL The ganuna-sitosterol gave a 98% match while beta-sitosterol gave 80% match. The fragmentation pattern (Figure B2-3.5) confIrmed the presence of a sitosterol type skeleton. The IR spectrum also verifies all the functional group peaks identifIed in the spectra. Thns, gamma-sitosterol was assumed to be the compound. 156 a HO m/z=414 clevage at a & b .. + ~ m/1F255 m/z= 141 + 'OH m/z= 17 m1z=85 + + m/z=329 CH C~ clevage at e 1-C2Ho ~C"i+ CH~ m1z=303 Figure 82-3.5: Major fragmentation pattern of sitosterol irrespective of the isomeric form. 157 B2.4.1 B2.4.2 CHAPTER B2-4 DISCUSSION Introduction In this chapter, the results for the isolation, partial purification and characterization of saponin B1 from Amadnmbe are discussed. Saponin The isolated saponin Bl present in Amadumbe tnbers was identified using TLC, IR and GC-MS techniques. The Rc value was calculated (0.64) to give a tentative identification of a sterol Rc value of compound Bl was similar to Rc value (0.65) of 5a-cholestan-3~? 01, the basic sterol from which other sterol structures are defined (Smith and Goad, 1975). The elucidation of this saponin was further identified by means of IR and GC-MS. Several researchers have investigated steroids in detail using GC-MS (Budzikiewicz et 01., 1964). Sterol fragmentation patterns are quite complex and it is possible that the typical fragmentation pattern of a specific structural unit appears as one set of sterol type and not another (Wretensjo, 2004). The molecular formula of compound BI was C29H500, based on the retention time of 22.54 (Tai et 0/,2004). The GC-MS spectrum showed a molecular-ion peak at 414 m1z, indicating a molecular weight of 414 (phuruengrat and Phaisansuthichol, 2006). Vibrations of functional groups of this compound were detected by infrared. The IR spectrum of compound B1 showed a band of Vrnax 3324 cm-t, which indicated the presence of -OH group without H-bonding. Furthermore, there were additional features in the spectra that identified the position and configuration of C-H and C=C stretching. 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(1998) Rice bifunctional a-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds. Biosci Biotechnol Biochem 62, 978-985. 180 SECTIONC EFFECTS OF INGESTED BETA-SITOSTEROL ON DIGESTIVE AND ABSORPTIVE ENZYMES IN RATS SUMMARY The purpose of this study was to investigate the effect of beta-sitosterol on intestinal disaceharidases and Na+/K+-AlPase activities in Sprague-Dawley rats. During the test period, food and water consumption, body weights and clinical signs were examined. Haematological changes included elevated alanine aminotransaminase (ALT) and aspartate aminotransaminase (AS}) levels. Histopathology revealed that the beta-sitosterol bad no apparent effect on the liver, kidneys or small intestine. The supplemented diet reduced Na+/K+-A1Pase and intestinal disaccharidases activities. 181 CHAPTERC-l LITERATURE REVIEW c.1.1 Introduction Plants are the only producers of phytosterols, which are cholesterol-like chemicals. The most common phytosterols are beta-sitosterol, campesterol and stigmasterol (Ling and Jones, 1995). Phytosterols are known for their ability to lower plasma cholesterol and there have been many studies reporting their hypocholesterolaemic effects (reviewed by Ling and Jones, 1995; Pollak and Kritchevsky, 1981). Epidemiological studies have also proposed that increased dietary phytosterol intake may decrease the risk of colon cancer in humans (Hirai et aI., 1986; Nair et al., 1984). In the previous sections, Amadumbe tubers were screened for the presence of anti nutrients. From the screened anti-nutrients, a-amylase inhIbitor and saponin were extracted, isolated and identified. The saponin was structurally elucidated as gamma sitosterol. Beta-sitosterol is a structural isomer of gamma-sitosterol and is a compound with many biological activities. Therefore, gamma-sitosterol would be the perfect anti nutrient compound for nutritional evaluation. Gamma-sitosterol was not commercially available and, because beta-sitosterol is the stereoisomer, it was decided to use beta sitosterol for the nutritional evaluation. A large body of research on the cholesterol lowering activity of beta-sitosterol has already been conducted; thus, the effects of beta sitosterol on digestive and absorptive enzymes were investigated in this study. '::.1.2.1 Stereochemical structure Sterols are necessary cell membrane components and are produced by both animals and plants. Plant cells consist of a nmnber of complex mixtures of sterols, whereas animals 182 C.1.2.2 and fungal cells contain one major sterol: cholesterol and ergosterol, respectively. More than 100 different types of phytosterols (plant sterols) have been identified (Morean et al., 2002). Seeds, nuts, fruits and vegetable oils are some of the different parts of a plant that contain significant amounts of plant sterols (Weihrauch and Gardner, 1978; Moreau et al., 2001). The phytosterol content of these vegetables and fruits is not influenced by intense processes such as boiling, bleaching and deodorizing since phytosterols are very stable compounds (Normen et aI., 1999; Bortolomeazzi, 2000). The human body is unable to produce plant sterols: thus, all plant sterols are regulated by dietary intake. Beta -sitosterol, stigmasterol and campesterol are the most abundant in plants (Rosenblum et al., 1993; Law 2000; Hicks and Moreau, 2001). Pbarmacochemistry and pharmacokinetics Researchers have been fascinated by the concept of a protein-mediated transport system responsible for intestinal uptake of cholesterol for more than a decade (Thwnhofer and Hauser, 1990). Identification of the molecular defects responsible for phytosterolemia (Berge et al., 2000; Lee et al., 200 I) and possible confirmation of the existence of a specific transport protein located in the brush border membrane mediating intestinal sterol absorption (Detmers et al., 2000; Hemandez et aI., 2000; Kramer et al., 2000) has shed new light on the cellular transport of cholesterol and plant sterols. Plasma phytosterol levels are generally very Iow compared to cholesterol levels in mammalian tissues. This is due primarily to poor absorption from the intestine and faster excretion from the liver (Ling and lones, 1995). Absorption of beta-sitosterol is also made difficult during its passage through the gut because these phytosterols are bound to the fibers of the plant (Bouic, 1998). The plant sterol mixture has very Iow solubility (Moghadasian, 2000): it is not soluble in either water or oil (Kim et aI., 2002). About five per cent of an ingested dose of supplemental beta-sitosterol is absorbed from the gastrointestinal tract and is transported via the portal circulation to the liver (Salen et al., 1970). Systemic circulation is 183 C.1.2.3 respoDSlble for transport of beta-sitosterol to other tissues in the body. Some beta sitosterol is glueoronidated in the liver and a portion is also metabolized to cholic acid and chenodeoxycholic acid (Marteau et al., 1980). Excretion is mainly via the biliary route. In an attempt to improve sterol solubility, plant sterols have been esterified with fatty acids in order to prodoce oil-soluble plant sterol esters (Mattson, 1964, U.S. Patent No. 5,502,045). Biological activities Beta-sitosterol offers a remarkable display of scientifically recognized benefits for major areas ofhea1th. Health benefits include: ? lowering cholesterol (Farquhar et al., 1956; Lees et ai, 1977); ? anti-diabetic properties (Ivorra, 1988); ? anti-tumor activities (Romero and Lichtenberger, 1990; Janezic and Rao, 1992); ? anti-bacterial properties (Hess et al., 1995; Padmaja et aI., 1993); ? anti-fungal abilities (Smania et al., 2003); ? anti-inflammatory predilection (Bouic et al., 2001); ? anti-atherogenecity activities (Moghadasian et aI., 1997); ? anti-ulcer properties(Jayaraj et al., 2003); and ? preventing cardiovascular events (Miettinen et al., 1990; Gylling and Miettinen, 1997). Human research already includes the treatment of hypercholesterolemia, benign prostatic hypertrophy (BPH) and rheumatoid arthritis with beta-sitosterol (pegel, 1997). Success in these areas, particularly with regard to cardiovascular diseases, may be attributed to the cholesterol-lowering effects ofplant sterols (Drexel et at., 1981; Miettinen et at., 2000). 184 Beta-sitosterol has been successfully used to lower cholesterol levels in humans, with almost no change in diet or exercise. Many scientific stodies have shown that, because beta-sitosterol and cholesterol are very similar in chemical composition, beta-sitosteriol interferes with cholesterol absorption by inlnbiting its absorption in the gut (Ling and Jones, 1995; Westrate and Meijer, 1998; Hallikainen and Uusitupa, 1999). This lowering of cholesterol helps to prevent the surplus rise in serum cholesterol from building up on the walls of the heart arteries. Bouic et al. (1996) reported on a series of in vivo and in vitro studies which clearly demonstrate that beta-sitosterol has immunomodulatory properties. Beta-sitosterol both improves an under-performing immune system to help fight viral and other infections and also corrects the underlying immune dysfunction of an overactive immune system as in autoimmune disorders such as arthritis. Rheumatoid arthritis, for example, is an inflammatory disease characterized by dysregulation of the immune system. B lymphocytes become overactive and secrete antibodies that destroy synovial tissue of the joint Beta-sitosterol and beta-sitosterol glucoside in combination have been shown to increase the levels ofT-Helper-l (TH1) cells, down-regulating antIbody production by B lymphocytes. The phytosterol mixture also decreased sections of pro-inflammatory cytokines by macrophages, thereby decreasing inflammation (Calpe-Berdiel et al., 2007). Sterols have been found to stimulate the body's immune system to fight the infections which disrupt the lives of HlV-positive patients (Bouic et al. 2001). Scientific reports have shown that CD4 lymphocyte counts can be maintained by beta-sitosterol and its glucoside, thereby slowing disease progression (Bouic et al., 1996). The studies also revealed an important decrease in IL-6 levels, possibly slowing viral replication rates in infected cells and thereby decreasing viral loads (Bouic, 1997). Studies show that beta-sitosterol is among the most successful treatments available for prostate enlargement (Buck et al., 1996; Klippel et al., 1997). The growth of human prostate cancer cells may be inlnbited by beta-sitosterol: this phytosterol apparently 185 C.1.3 Cl.3.! replaces some of the cholesterol in the cell membrane, which ultimately triggers the relaying of an instruction to the cells not to divide (Awad et al., 2000). Thus, dietary plant sterols also have possible anti-cancer effects. Beta-sitosterol may offer protection against breast cancer by stimulating apoptosis and by inhibiting tumor growth in cancer tissue (Awad and Fink, 2000). Epidemiological studies have shown positive association between higher dietary phytosterol intake and populations at lower risk for colonic cancer (Hirai et al., 1986). Experimental studies by Raicht et al., (1980) have shown that rats fed a diet containing 0.3 percent beta-sitosterol had a significantly lower incidence of tumours when given methylnitrosourea (MNU), a chemical carcinogen, than those on diets without beta-sitosterol supplement. The toxic potential of a test article may be considerably affected by several factors such as administration route, dosing form and the experimental conditions (Dain and Jaffe, 1988; Dethloff et aI., 1996; Kim et aI., 2001). With regard to toxicity, no obvious side effects of phytosterols have been observed in studies to date, except in individual with phytosterolemia, an inherited lipid disorder (Ling and Jones, 1995). Beta-sitosterol has been fed to rats in dosages of between 0.5 and 5 mg kg-1 (Young et al., 2004; Malini and Vanithakumari, 1990, 1992). Kim et al (2002) investigated subchronic toxicity of plant sterol ester by administering 1000, 3000 and 9000 mg kg-1day-l to rats . Based on their results, 9000 mg kg-lday-l was considered to be the toxic dose since it resulted in the suppression ofbody weight gain in both sexes and male cardiomyopathy. Digestive enzymes Disaccharidases The nutritional building blocks derived from digestion are used by metabolic enzymes in every cell, tissue and organ of the body to restore damage and decay, to combat and overcome disease, to repair wounds and to maintain overall health and well-being. 186 Digestive enzymes do the all important work of hydrolyzing food into the nutritional components required to build and maintain health. Disaccharides (sucrose, lactose and maltose) are important sources of metabolic energy (Dilworth et al. 2005). Disaccharidases are integral enzymes of the small intestinal brush border membrane responsible for the hydrolysis of carbohydrates which must occur before monosaccharides can be absorbed (Lorenzsonn et al., 1987; Re et aI., 2006). These enzymes are sucrase (BC 3.2.1.26), lactase (BC 3.2.1.23) and maltase (BC 3.2.1.20) respectively. The end-products (glucose and fructose, galactose) resulting from the activities of these enzymes are actively translocated from the intestine to the blood by ATPases (Dilworth et al., 2005). Although the factors controlling the development of intestinal disaccharidases are not known (HeIZenberg and Rerzenberg, 1959), it appears that the intestinal dissacharidases of mammals develop in a way that allows the utilization of the carbohydrates that the animal is likely to encounter under natural feeding conditions (Siddons, 1969). Thns, dissacharidase activities appear to be broadly correlated to dietary habits (Vonk and Western, 1985). Food influences the brush border enzyme activity in rats and is also necessary for the maintenance ofnormal mucosal stability (Yamada et al., 1981; Rolt and Yeh, 1992). Thus, the activities of specific enzymes are reduced when their substrates are sporadic or absent from an animal's diets (Zarling and Mobarhan, 1987; Rivera-Sagredo et aI., 1992). On the other hand, studies suggest that carbohydrate intake increase dissacharidase activities (Shinohara et al., 1986; Samulitis-Dos et aI., 1992). The overall digestive capacity of an animal is sensitive to changes in the concentrations of intestinal enzymes. These changes may affect the nutritional status of the animal, as well as the functioning of other systems, including neurological, renal, cardiac and pulmonary systems (Lee et al.,2003). 187 Intestinal disease may be determined by the activities ofdisaccharidases. Greater enzyme activity does not have any known clinical significance, but decreased enzyme activity results in a digestive defect of disaccharide, which, clinically, may result in osmotic diarrhea, crampy abdominal pain or gaseousness (He et ai, 2006). C.1.3.2 ATPase ATPases are a group of enzymes that catalyze the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and a free phosphate ion (Figure Cl-I). Energy is released from this dephosphorylation reaction and is used to drive other chemical reactions. l' I'! OHOH o 0 11 I -11-DHzC 1;1 ==='~ _0 _0 .<--__ ___..1;1 Figure Cl-I: Hydrolysis of ATP to ADP, the fundamental mode of energy exchange in biological systems (Moyna, 1999) Some ATPases are basic membrane proteins and transport solutes across the membrane and are therefore called transmembrane ATPases. Sodium-potassium exchanger is a primary example of a transmembrane ATPase which establishes the ionic concentration balance that maintains the cell potential (Figure CI-2) [Skou, 1957]. Sodium-potassium ATPase (Na+/K +- ATPase) is a wide-spread membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K + levels and fluid volume. Sodium- 188 potassium ATPase creates concentration gradients of sodium and potassium ions across the plasma membrane of animal cells. The gradients provide the somce of energy for membrane transport of substances. Ion gradients play an essential part in the flow of metabolites: for example, the basic glucose transport in intestinal enterocytes. Prolonged flow will dissipate these gradients, making them less efficient. Thus, it is essential to rebuild these gradients. Extracertu/ar Fluid Intracellular Fluid Sodium (Na'): 145 mmoUL "-QC?: -S oL--L..-"-__ 0.25 0' .2 0.05 ~i 0.15 ?i Figure C3-9 a: Sucrase 209 .'" ... .., ., ... Figure C3-9 b: Maltase .... ?.'" Figure 0-9 c: Lactase 210 CHAPTERC-4 DISCUSSION C.4.1 Introduction This section of the study was conducted to investigate the effects ofbeta-sitosterol on the digestive and absorptive enzymes of Sprague-Dawley rats. Results obtained for the nutritional evaluation are discussed in this chapter. C.4.2 Experimental test The rats were in good health throughout the study: no mortality was observed in any of the groups tested with beta-sitosterol. This is in agreement with studies conducted on phytosterols. Similar studies have consistently demonstrated a lack of toxicity in animals and humans fed beta-sitosterol., except for individuals with an extremely rare genetic condition, sitostero1aemia (Malini and Vanithakumari, 1990; Hicks and Morean, 2001). Plasma phytosterol levels are generally very low in mammalian tissues. Poor absorption from the intestine and quicker excretion from the liver, compared to that of cholesterol, are responsible for these low levels (Ling and Jones, 1995). Intestinal absorption of total dietary beta-sitosterol consumed is negligible in mammals. Animal studies have demonstrated that this absorption should possibly be as little as five percent (Borgstrom, 1968; Gould, 1955). In rats, the absorption of beta-sitosterol is about four percent (Sanders et al., 2000). A reason for the low absorption of plant sterols might be that plant sterols are poorly esterified, possibly due to the low affinity of ACAT (acyl-coenzyme A cholesterol acyltransferase) for these components (Bhattacharyya, 1981; Ntauios et al., 1998). Within the enterocyte, free cholesterol is is esterified by ACAT, incorporated into chylomicrons, which are subsequently excreted into the circulation and converted into a chylomicron-remnant by the action of lipoprotein lipase (Kwiterovich, 2000). Absorption 212 of free cholesterol depends on mixed micelles: mixtures of free cholesterol, mono- and diacylglycerols, fatty acids, phospholipids and bile salts (De Jong et al., 2003). Evidence has been presented to support the view that cholesterol absorption would take place only in the presence of fat. It has been demonstrated that the absorption of cholesterol is made possible by fat. The fatty acid component of fat is the active factor for this absorption (Kim and Ivy, 1952; Swell et al., 1956). The most efficient fatty acid promoting cholesterol absorption has been reported to be oleic acid (Swell et al., 1956). Cholesterol is structurally similar to beta-sitosterol. Consequently, the solubility of beta-sitosterol was enhanced by esterifying it with oleic acid. The results in this study showed that beta sitosterol became active in the rats when 0.01 mg kg-I day-I of oleic acid was added to the beta-sitosterol. Changes in the physiological state of mammals can alter the concentration of a number of organic constituents in the blood serum: for example, glucose, protein, cholesterol and liver enzymes (Malini and Vanithakumari, 1990). Triacylglycerol levels, which are independent risk factors for cardiovascular problems (Wierzbicki and Mikhailidis, 2002), showed a significant decrease in levels for rats fed beta-sitosterol compared with rats fed oleic acid. Similar effects have been observed in cells incubated with oleic acid; oleic acid increased both the intracellular pool and biosynthesis of triacylglycerols (Pullinger et aI., 1989; Ellsworth et al., 1986). The potential target for the influence of oleic acid is in the assembly of the apoB-containing (apolipoprotein B) lipoproteins. This occur in regions of the ER with hig1I diacylglycerol:acyltransferase activity that has a hig1I capacity for the formation of triacylglycerol (Boren et aI., 1990). Oleic acid could be nsed to manipulate important steps in the assembly process (Boren et aI., 1993). The decreased level observed in beta-sitosterol fed rats is in line with the known hypercholesterolemic activity of beta-sitosterol (Duivenvoorden et al., 2006). A slig1It decrease in serum protein level was observed in the group receiving beta sitosterol in its diet. The slig1It change in the serum protein levels may have resulted from altered rates of anabolism and catabolism (Malinia and Vanithakumari, 1990). When compared to the two control groups, serum cholesterol was slig1Itly, but not 213 significantly, decreased after beta-sitosterol treatment. Earlier reports have shown that beta-sitosterol is a potent inhibitor of dietary and serum cholesterol in rats (Gould, 1955), mice (Behar and Anthony, 1955), rabbits (Bhattacharyya and Lopez, 1979) and dogs (Shipley et al., 1958). Therefore, in the present investigation, the observed decrease in sennn cholesterol concentration appears to be due to the inherent hypocholesterolemic effect of beta-sitosterol. An increased sample size would be necessary to increase the inhibitory effect on cholesterol. Owing to poor solubility and bioavailability of phytosterols, the lowering effect on sennn cholesterol of phytosterols is not consistent. High dosages of between 25 and 50 gld are required for efficacy (Moreau et al., 2002). Aminotransferase enzymes, ALT and AST, are largely used in the assessment of liver damage (Al-Habori et aI., 2002; Dobbs et al., 2003). These enzymes can be measured in sennn since membrane damage to the liver releases the enzymes into circulation. In the sennn biochemical analysis, the most notable results were significantly elevated ALT and AST. Such a significant increase in enzymatic activity of sennn ALT and AST reveals a very important pathological change in cell-membrane permeability or hepatic-cell rupture (Benjamin, 1978). This rise in liver enzyme activity is not necessarily an indication of the liver's ability to synthesize the enzymes. Instead, it signifies a loss of material from damaged hepatocytes (Woodman, 1980). It is generally assumed that an increase of these enzyme activities reflects active inflammation and necrosis of hepatic cells. The levels of alkaline phosphatase remained fairly stable in this investigation and, therefore, do not appear to confirm the early stages of viral infection. However, it should be borne in mind that increases in alkaline phosphatase levels are not as sensitive an indicator of hepatic viral infection as are elevated ALT and AST (Gopal and Rosen, 2000). The significantly elevated ALT and AST levels were not correlated to any observable clinical changes in the livers. Clinical observations included liver weights, as well as macroscopic and microscopic histological examinations of the livers and kidneys. A 214 significant increase was observed in the liver and kidney weights in the group receiving oleic acid, but, macroscopically and microscopically, all livers and kidneys appeared normal. With viral infections, ALT and AST levels are elevated even before the clinical signs and symptoms of disease occur. ALT values increased 13.4 and 17.9 times respectively for distilled water and oleic acid control groups, whereas ALT levels increased 4.6 and 6.0 times respectively for the two control groups. This is in agreement with Jobnston (1999), who reported that in typical viral or toxic liver injury, the serum ALT levels rise more than the AST value. Observations, using an electron-microscope, might have revealed damage to hepatocytes which could have caused elevated ALT and AST levels. Oxidative stress at the sub-organeIIe (mitochondria and macrosome) level could have led to build up of free radicals that could have caused increased ALT and AST levels (Chen et aI., 1998; i~en et al., 2005; Balkan et aI., 2002). Silva et al., (2004) have observed and increase in AST and ALT activities in the liver of bullfrog oil-treated animals compared to the control. Bullfrog oil is a MUFA-rich (monounsaturated fatty acid) animal derived oil. MUFA account for 55% of total fatty acids, consisting of 38 % oleic acid. Their results indicate that the increase in oxidative stress (lipid peroxidation and catalase activity) in the liver ofbullfrog oil-treated animals promoted damage to liver cells, which led to an increase of AST and ALT activities in the liver (Silva et al., 2004). According to this study beta-sitosterol administration can improve the stress if obtained by oleic acid. White blood cells (WBC) are generaIIy crucial in fighting any infection (SchaIm et al., 1975). In this study, there was a significant decrease in the WBC count for the two control groups, which might imply reduction in the ability to respond to early infection. However, the normal WBC levels for the group fed beta-sitosterol were significantly increased in comparison with the control rats fed distiIIed water and oleic acid. This might indicate a boost to the immune system. The group receiving beta-sitosterol also showed significantly lower AST and ALT levels relative to both control groups. Bouic et al. (1996) demonstrated that beta-sitosterol may have immunomoduIatory properties and that even though this phytosterol is poorly absorbed and not synthesized in the human body, daily intake is needed to sustain an optimal immune response. Therefore, these results might 215 indicate that beta-sitosterol boosts lhe production of WBC and could also have a possible hepatoprotective function (Banskota et al., 2000). Gross inspection of lhe livers, kidneys and small intestines revealed hardly any visual lesions attributable to sterol treatment There were no remarkable differences in variability of the histopalhological parameters in the groups studied. All lhe groups showed normal cellular architecture, with distinct hepatic cells, sinusoidal spaces and periacinar veins. Glomeruli, Bowman's space, proximal convoluted and distal convoluted tubules appeared normal for alllhe kidneys. The availability of the surface area for various aspects of digestion and absorption in the duodenUlll, jejunum and ileum is very important for proper functioning of the small intestine. Following lhe histopalhological examination, no adverse changes in villus height or mucosal surface area were discovered in the dnodenum or ileum and, therefore, no apparent effects on the functioning of the intestine were observed. None of lhe minor changes appeared to be related to the exposure to diets containing beta-sitosterol supplement. This suggests that beta-sitosterol is devoid of any toxic complications or side effects at the dose tested. These findings are in agreement with previous reports that the liver and kidneys did not show any adverse effects after long-term exposure to oral administration ofbeta-sitosterol in rat, rabbit and dog models (Swell et a/., 1956; Shipley et al., 1958; Malini and Vanithakumari, 1990; Hepburn et al., 1999). Intestinal ATPase activity was significantly increased in the control group receiving oleic acid in their diet, in both intestinal locations. The Na+/K+-ATPase showed less significantly elevated activities after beta-sitosterol intake, compared with rats in the control group receiving the oleic acid. Information on the effect of beta-sitosterol on ATPase activity is scanty in literature. Takabashi et al (2006) showed that ATPase activity of ABACI (ATP-binding cassette protein AI) was reduced in a dose-dependent manner by the addition of cholesterol, decreasing by 25 percent in the presence of 20 percent cholesterol. Beta-sitosterol, which does not have a double bond in the acyl chain as cholesterol, showed a similar inhibitory effect as cholesterol. ATPase activity may be 216 decreased by sitosterols as a result of their affect on membrane fluidity (Silva et al., 2005). Increased Na+JK+-ATPase activity generates an inward sodium ion gradient that leads to increased glucose translocation across the cell membrane (Omoruyi, 1991). A reduction in ATPase activity can result in lowered glucose translocation from the intestine into blood. This could be useful in the treatment of diabetics (McAnuff et al., 2005). When rats fed with beta-sitosterol were compared with rats fed the oleic-acid and/or distilled water control diet, a significant inhibitory change was observed. Upper intestinal sucrase and maltase and both proximal and distal intestinal lactase activity showed a significant inlnbitory effect (p>O.05). The dissemination of disaccharidase activities among mammals is extremely diverse (Martinez del Rio and Stevens, 1988). The distnbution pattem of the disaccharidases along the small intestine is also significantly influenced by the locus of disaccharide hydrolysis because the location of optimum activity presumably reflects the site of absorption. A number of mammals have showed this inconsistent distrIbution of disaccharidase activity along the small intestine (Dahlqvist, 1964; Malhotra and Philip, 1964, 1965). This non-uuiform distribution of disaccharidase activity along the small intestine was also observed in this study. The modified changes in enzyme activity of the small intestine depend on various exogenous factors (McCarthy et aI., 1980). These disaccharide mucosal enzymes are physiologically necessary in the digestion of food consumed for assimilation. They provide essential nutrients for maintenance and rapid growth (Lee et aI., 2003). Reduced disaccharidase activity is indicated by reduced blood glucose levels from less absorbable glucose formed from carbohydrate digestion (McAnuff et aI., 2005). There are other factors that can influence disaccharidase activities, such as obesity and age (Flores et aI., 1990), hormones (Raul et al., 1984) and a decrease of luminal proteases (Zarling and Mobarhan, 1987). Thus, with a decrease in the blood-glucose levels of the rats treated with beta-sitosterol, the decreasing effect on the activities of the disaccharidase and ATPase could suggest the value of beta-sitosterol an as anti-diabetic (hypoglycemic) agent. 217 CHAYfERC-5 CONCLUSION C.S.! Beta-sitosterol was administered to rats over a period of 14 days. No evidence of toxicity was observed in the rats given daily oral dietary supplements containing beta-sitosterol. No gross or microscopic alterations of any tissues were observed. There was no histological evidence of deposition of the plant sterol. 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Vank RI. and Western J.R.R (1985) Comparative biochemistry and physiology of enzymatic digestion. Academic Press, London. pp 255-295. Wierzbicki AS. and Mikhailidis HP. (2002) Beyond LDL-C - the importance ofraising HDL-C. Current Med Res Opin 18, 36-44. Weihrauch J.L. and Gardner J.M (1978) Sterol content of foods of plant origin. JAm Biabetes Assoc 73, 39-47. Weststrate JA and Meijer GW. (1998) Plant sterol-enriched margarines and reduction of plasma total- and LDL-cholesterol concentrations in normocholesterolaemic and mildly hypercholesterolaemic subjects. Eur J Clin Nutr 52,334-343. Woodman DD. (1980) Assessment of hepatic function and damage in animal species. A review of the current approach of the academic, governmental and industrial institutions represented by the Animal Clinical Chemistry Association. J Appl Toxicol, 8,249-254. Yamada K., Bustamante S. and Koldovsky O. (1981) Dietary-induced rapid increase of ratjejenul sucrase and lactase activity in all regions of villus. FEBS Letters 129, 89-92. Zading EJ. and Mobarhan S. (1987) Effect of restricting a balanced diet on rat intestinal disaccharidase activity and intestinal architecture. Journal of Laboratory and Clinical Medicine 109, 556-559. 233 CHAPTER 2 GENERAL CONCLUSION 2.1 Introduction The mass production of highly refined foods has tended to overshadow traditional foods which in many ways are more nutritious. In search for solutions to problems of food insecurity in developing countries, there is need to re-examine the role of these traditional foods. Amadumbe is widely grown in the sub-tropical parts of South Africa. The locally developed cu1tivars are commonly used as staple foods in the Kwazulu-Natal province, South Africa. When any crop is being considered as a food source the nutritional value is of utmost importance. The nutritional and anti-nutritional evaluation of unprocessed and processed Arnadumbe tubers revealed the following: 2.2 Nutritional and anti-nutritional evaluation Proximate analysis showed Arnadumbe to be highly nutritious with high carbohydrate, adequate protein and low lipid content Amadwnbe was also shown to contain essential fatty acids in the form of linoleic and linolenic acid. The tubers are generally low in mineral content except for potassinm and magnesium. Even though anti-nutritional substances were found in unprocessed Arnadumbe, domestic processing (boiling, frying, roasting), were observed to effectively reduce the content of the anti-nutrients Boiling was the most effective method to decrease the levels of anti nutrients. Zululand Colocasia esculenta can therefore be used as food material for humans but tubers should be processed properly to not pose a long-term health problem in humans. 234 2.3 Cbaracterization and nntritional evaluation ofselected anti-nutrients in Amadumbe The characterization of two anti-nutients a-amylase inhibitors and gamma-sitosterol highlighted some of the benefits of the consumption of Amadumbe. The a-amylase inhibitors present in the Amadumbe tubers showed inlnbitory activity against mammalian amylases and could therefore cause a marked decrease in the availability of digested starch. This may be beneficial in the management ofdiabetes mellitus and obesity. Gamma-sitosterol was also isolated from Amadumbe tubers. It is an isomer of beta sitosterol which is a highly active biological compound. The nutritional evaluation of beta-sitosterol has shown that by lowering the activity of disaccharidases, it could cause a decrease in glucose concentration in the blood of rats. Therefore the gamma-sitosterol present in these tubers could possibly be valuable by reducing blood sugar levels in diabetics and obesity. The death rate among adults in South Africa is increasing. AIDS-related illnesses are not likely to be the entire reason for the increase in death rates. With reported increase in urbanization and the associated changes in life-style, incidences of obesity and diabetes could be additional factors. The results of this study do suggest that even though Amadumbe is a neglected crop in South Africa, it is a highly nutritional crop, the consumption of which could be beneficial to diabetic and hypertensive patients. 235 2.4 Suggestions for future work Further studies on Amadumbe are suggested on these lines 1. To extract and partially characterize other anti-nutritional factors in Amadumbe tubers. 2. Studies on the properties of these anti-nutrients and their effect on the human health. 3. Investigate the extent to which a-amylase inhibitors are capable of interfering with the digestive process ofhumans on consumption of Amadumbe. 4. Extract beta-sitosterol and/or gamma-sitosterol from Amadumbe to feed rats and do full investigation on effect of the beta-sitosterol on glucose absorption which could be beneficial in diabetes and obesity. 236 AppendixA PREPARATION OF REAGENTS A.A.l Anthrone reagent 1 g of Anthrone was dissolved in 500 ml of72% H2S04 . A.A.8 Indicator 0.0172 gbromocresol green and 0.0078 g of methyl red were added to 25 ml of ethanol. A.A.3 Dam's reagent Preparation of pyridine dibromide solution. This was prepared by the addition of 2.06 ml pyridine in 5 ml glacial acetic acid to another solution of L 85 ml concentrated sulphuric acid in 5 ml glacial acetic acid and was cooled To this mixture of 0.63 ml ofbromide diluted with 500 ml glacial acetic acid. Potassium iodide solution (10%) 10 g of K.I dissolved in 100 ml of distilled water. Starch indicator solution of 1 g soluble starch dissolved in potassium chloride solution which was allowed to boil and cool. A.A.2 0.05 M Tris-Hydrochloric Acid buffer pH 8.6 6.05 g Tris (hydroxyl methyl) amino methane was dissolved in a litre of distilled water and labelled'A'.4.3 ml of Hel was diluted to 1 litre with distilled water and labelled solution 'B'. 50 ml of A was mixed with 5 ml ofB and made up to 200 ml with distilled water. The pH was adjusted to 8.6 with 4.0 MNaOH solution or dilute Hel solution. 237 A.A.3 Enzyme trypsin 10 mg bovine-pancreas trypsin type I was dissolved in 0.001 M HCl. The enzyme was stable in a refrigerator for two weeks. 100 mg protease was dissolved in 1 litre of tris-HCl buffer, pH 8.0, (i.e. a-ehymotrypsin type vii from bovine pancreas, protease type xiv from Streptomyces Griseus, proteinase type xviii from Rhizopus species and protease type xiii fungal from Aspergillus SaitOI). A.A.4 0.001 M BAPNA (Benzoyl-DL-arginine-p-nitroaniline) 43.5 mg of benzoyl-DL-arginine-p-nitroaniline (BAPNA) was dissolved in I ml of dimethyl sulfoxide (DMSO) solution. This was diluted to 100 ml with 0.05 M tris-HCl buffer pH 8.6, containing 0.02 M calcium chloride. Care was taken to dissolve all BAPNA in the DMSO, as the presence of any crystals causes precipitation to occur on standing. A constant temperature was maintained throughout preparation. A.A.5 Phosphate buffer pH 6.9 18.73 g of sodium dihydrogen orthophosphate and 8.15 g of disodium hydrogen orthophosphate were mixed, dissolved together in a litre of distilled water and the pH was adjusted to 6.9. A.A.6 Soluble starch 1 gofsoluble starch was dissolved in 100 ml ofphosphate buffer. A.A.7 Dinirrosalicylic acid (DNS) 1 g of 3.5 dinitrosalicylic acid was dissolved in 20 ml of 2 M NaOH and 50 ml of distilled water. 30 g ofRochelle salt was added and made up to 100 ml with distilled water. 238 A.A.8 0.04 M Sodium borate buffer pH 8 2.473 g of boric acid was dissolved in 1000 ml distilled water. 2.012 g of sodium borate dissolved in 250 ml of distilled water. Then 166.67 ml of sodium borate was mixed together with 1000 ml ofboric acid. A.A.9 ADA 100 g of dextrose; 68.296 g of 0.065 M citric acid and 124.95 g of 0.085 ml trisodium citrate were dissolved in 5000 ml of distilled water. A.A.10 Washing buffer (pH 6.5) 32.77 g of 0.113 M NaCl; 3.741 g of 4.3 mM K2HP04; 14.64 g of 24.4 mM NaH2P04; 5.45 g of 5.5 mM glucose and 1.86 g of 1 mM EDTA were dissolved in 5000 ml of distilled water. AA.11 Resuspending buffer (pH 7.4) (i) 41 ml 0.03 M HCl was added into 50 ml of0.03 M tris amminomethane and the solution was made up to a 100 ml with distilled water. (ii) 8.12 g of 0.14 MofNaCl and 0.99 g of 0.005 M of glucose was dissolved in (i). A.A.9 0.1 M Ferric ammonium sulphate (FeNH.(S04h) in 0.1 M HCI 8.3 ml of concentrated HCl was diluted to 0.1 M by bringing the concentrated acid to I L with distilled water. Ferric ammonium sulphate was made by dissolving 4.8 g of the dodecahydrate salt Fe~(S04)z .12 H20 in 100 ml of the 0.1 M HCI. The resulting solution was pale yellow. AA9 0.008 M Potassium ferricyanide (K3Fe(CN)6) 0.26 g of potassium ferricyanide was dissolved in 100 ml of distilled water. The resulting solution was yellow. A.A.10 Vanillin solution 800 mg of vanillin was dissolved in 10 ml of 99.5 per cent ethanol. 239 A.A.ll 0.1 M Orthophosphorieletbanol medium 6.78 mI of orthophosphoric acid was dissolved in I litre of distilled water and 250 mI of ethanol was added to the orthophosphoric acid. AB.l Phosphate buffer 18.74 g of sodium dihydrogen orthophosphate ( ) and 8.16 g of disodium hydrogen orthophosphate ( ) were mixed and dissolved together in a litre of distilled water. pH was adjusted to 6.9. AB.2 Soluble starch I g of soluble starch was dissolved in 100 mI ofphosphate buffer. AB.3 Dinitrosalicylic acid 1 g of 3.5 dinitrosalicylic acid was dissolved in 20 mI of 2 M NaOH and 50 mI of distilled water. 30 g of Rochelle salt was added and the resulting mixture was made up to 100 ml with distilled water. AB.4 Tris-HO 0.72 mI HCl was added to 10 mI of distilled water. 6.06 g ofTris was dissolved in 200 mI of distilled water and 9.75 ml of HCl. The resulting mixture was made up to 250 m1, then up to 1000 mI with distilled water and the pH was adjusted to 7.5. AB.5 0.4 M NaO phosphate buffer 23.38 g ofNael was dissolved in 500 mI ofphosphate buffer. AB.6 G-l00 Sephadex 109 of Sephadex was dissolved in 500 mI ofphosphate buffer. 240 A.C.l 10 per cent buffered formalin 1.75 g of sodium hydrogen orthophosphate (NazHP~HzO) and 3.25g of di-sodium hydrogen orthophosphate (NazHP04) was dissolved in 25 ml ofboiling water. 50 ml of 40 per cent formalin was added and the resulting mixture was made up to 400 ml with distilled water. 241 AppendixB DETAILS OF MEmOOOLOGY BA1 Proximate analysis (AOAC, 1990) BA1.1 Ash determination Volatile organic matter was driven off when 2.5 g of the sample was ignited and kept at 6000 C for six homs in an electric furnace. The residue was quantitated and the mass of the inorganic matter over the mass of the organic matter was noted as percentage ash. BA1.2 Moisture determination 50 g of the sample was transferred to previously weighed and dried crucibles, then dried in a thermostatically controlled oven at 1l0? C for 24 hOUTS. Samples were then removed, cooled in desiccators and weighed. The final weight over the initial weight was recorded as the percentage ofmoisture. BAl.3 Crude fat determination 0.5 g of dry samples was pre-extracted with 25 ml of petroleum ether for 50 minutes with an automatic soxhlet system (Soxtec HT-6, Tacater AB, Hoganas). The solvent in the extraction cups was evaporated overnight in an oven (700 C) and weighed to calculate the fat percentage. BAl.4 Soluble carbohydrate determination [Hansen and Meller (1975)] Extraction and determination of soluble carbohydrates were performed according to the methods of Hansen and Maller (1975). One gram of each sample was quantitatively transferred into bmettes stuffed with glass wool. These bmettes had been previously set up as shown in Figure B-1. The samples were wet with 2 ml of 80 per cent ethanol each and stirred to remove air bubbles in order to avoid channel formation. 242 Liquid surfaces were fonned with ethanol. The air spaces were created by inserting a stopper of glass wool as shown in Figure 8-1. The air space was to prevent diffusion of the carbohydrate into the solvent. The soluble carbohydrates were percolated from the sample. Burette Solvent Glass wool AirSpace Liquid surface Test sample Glass wool Stopper Figure B-1: Burette packaging for starch percolation After ethanol percolation, the residues containing starch were further percolated with 25 ml of 35 per cent perchloric acid each. The residues were thoroughly mixed with 2 ml perchloric acid first, which resulted in their swelling. This was also percolated at the rate of 1.5 ml hour'! . 2 ml of the test solution containing starch and glucose was pipetted into test tubes and kept at 0? C. la ml of anthrone reagent, which had been cooled to 0? C, was added to the 2 ml test solution. The reaction was thoroughly shaken and heated for exactly la minutes in a 100? C water bath. Thereafter, the test tubes were cooled to 0? C and the absorbance was read at 630 om against 2 ml of 30 per cent perchloric acid, 10 ml of anthrone reagent for starch, 2 ml of 80 per cent ethanol and la ml of anthrone reagent for soluble sugars. The amounts of starch and soluble 243 sugars were estimated from standard graphs, prepared using potato starch (starch) and glucose (soluble sugar). B.A.l.S Protein determination (Kjeldahl method of nitrogen determination) B.A.l.S.l Digestion I g of the dried and ground sample was transferred into a digestion flask. One Kjeldahl tablet and IQ ml of concentrated H2S04 were added to each flask. The blank was prepared using the tablet and concentrated H2S04. The mixture was cautiously heated on the digestion stand in a fume cupboard until the acid became clear. When the digestion was completed, samples were removed to cool, 2 ml of distilled water was added and the samples were cooled again. B.A.l.S.2 Distillation 5 ml of the sample and 20 ml ofNaOH solution were added to the digestion flask. A conical flask was prepared by adding 10 ml of saturated boric acid and 6 drops of mixed indicator (0.0172 g bromocresol green and 0.0078 g methyl red) so that the condenser tip was immersed. Distillation was completed and 30 ml of the distillate was collected in the conical flask. The tip of the condenser was removed from the conical flask. B.A.l.S.3 Titration The distillate was titrated with 0.05 M Hel to a pink endpoint. The crude protein content of the Amadumbe tubers was calculated from the results, using a factor of 6.25 to convert the amount of nitrogen to crude protein. B.A.l.SA Fatty acid determination lA.l.5.4.l Lipid extraction [Bligh and Dyer (1959?) 50 g of Amadumbe were extracted with ISO ml of methanol-chloroform (2:1, v/v) mixture overnight in the oven shaker. The homogenate was filtered and the filter residue was re extracted overnight with a mixture of methanol-chloroform (2: I, v/v) and 40 ml of distilled 244 water. The homogenate was filtered and the filter residue was then washed with 75 ml of methanol-chloroform (2:1, v/v). The filtrates were combined in the separating funnel with 125 ml of chloroform and 145 ml of water and phases allowed to separate. The chloroform layer was then collected and diluted with benzene and concentrated in vacuo. The residual lipids were immediately dissolved in 25 ml of chloroform-methanol (1:1, v/v) mixture. B.A.I.S.4.2 Hexane extraction 15 g of dried Amadumbe was dissolved in 90 ml of hexane. The mixture was stirred occasionally for 15 minutes. Solid particles were filtered using rough filter paper and the extract was filtered again using fine filter paper with 0.45 lIlI1 pores. Hexane was evaporated at room temperature to yield the plant oil. B.A.I.S.4.3 Iodine value 5 mg lipid was dissolved in 5 ml chloroform and to this solution 5 ml ofDaIn's reagent was added in a 50 ml Erleumeyer flask. The solution was mixed and left at room temperature in the dark for 15 minutes. 0.5 ml of potassium iodide was added to this mixture with 0.5 ml of water and few drops of starch indicator. A standard thiosulfate solution was used to titrate the liberated iodine to the endpoint colour of milky. A blank was titrated containing 5 ml of chloroform only. B.A.I.S.4.4 Column Chromatography 20 g of silicic acid was preheated in the oven at 120?C. A slurry of the gel was then prepared with 35 ml chloroform in a beaker and poured into a 48x2.l5 cm chromatography tube,the stopcock was openend slowly to dislodge the air bubbles which result in settling of the column. The solvent level was allowed to drop to the top of silicic acid; the bed was washedwith 2 column volumes of 35 ml chloroform. The Amadumbe exract was added in the column at the top of the solvent and the elution of the column was done at the flow rate of 3 ml/min with 175 ml chloroform., 10 column volumes, 40 column volumes with 700 ml acetone and 10 column volumes with 175 ml methanoL 245 B.A.l.5.4.5 Liquid Chromatography Mass-Spectroscopy The extracts of Amadumbe extracted with hexane and methanol-chloroform mixture were dissolved in methanol and the dichloromethane. 200 J-Ll of this solution was then made up to 2 ml using the mixture of methanol and water. The analysis of the fatty acids was conducted under the following conditions by LCMS whereby the mass spectrometer conditions were kept in the capillary voltage of 3500 kV, cone voltage, ISO kV, desolvation temperature 250 DC and desolvation gas flow rate 300 DC. The solvents used was methanol and water at the flow rate of 0.035 ml min? l . The dissolved extracts were then injected in LCMS at an injection volume of5 J-Ll. B.A.2 Determination of anti-nutrients B.A.2.1 Trypsin inhibitor [Smith et aL (1980?) Ig Amadumbe powder was extracted by homogenizing it in 20 g/litre of NaCl at the ratio 1:10 (w/v). The homogenate was stirred for 24 hours at room temperature, passed through a layer of cheese cloth and centrifuged at 9000 rpm for 15 minutes. The supernatant was passed through glass wool to remove any floatiog lipid materials. B.A.2.1.1 Enzyme inhibitor assay The following additions were pipetted into a series of 10 ml test tubes containing: ? reagent blank: 2 ml of distilled water (test tube a); ? standard trypsin: 2 ml of standard trypsin solution, 2 ml ofwater distilled water (test tube b); ? sample blanks: 1 ml of diluted sample extract, 1 ml of distilled water (test tube c); ? test samples: 1 ml of diluted sample extracts, 1 ml of distilled water, 2 ml of standard trypsin solution (test tube d). Afler mixing the solutions and heating them to 370 C for 10 minutes,S ml of BAPNA solution (previously warmed to 370 C) was pipetted into each test tube and the resulting solution was mixed. Afler an incubation of exactly 10 minutes at 370 C, the reaction was stopped by adding 1 ml of 39 per cent (v/v) acetic acid. The absorbance was measured at 246 410 DID and the colour remained stable for several hours. Solutions of trypsin and BAPNA were prepared as descnbed by Kakade et at (1974). This BAPNA is an artificial substrate that becomes yellow when it reacts with the trypsin, thus revealing the non-inlubited trypsin (Kakade et at., 1974). B.A.2.1.2 Calculation The change in absorbance (At), owing to trypsin inlubition ml-! diluted sample extract is (AJ, - Aa) - (A.! - A,), where the subscripts refer to tubes (a) - (d) referred to above. The percentage inhibition in each sample tube is given by 100 At/(AJ, - Aa). If this is less than 40 per cent or more than 60 per cent, the assay must be repeated to provide a mOTe suitable dilution (D) of the sample suspension. The trypsin inlubitor activity (TIA) was calculated in tenus ofmgpure trypsin g'! sample as weighed (mg g'!): 2.632?D ?At TIA = S mg pure trypsin inhibited g'! sample B.A.2.2 Amylase inhibitor [Bernfeld (1955)) 50 g of dried Amadumbe powder was homogenized and defatted with hexane. The samples were extracted with distilled water containing 1 per cent polyvinylpolypyrrolidone (pVP). The resulting crude extract was centrifuged at 10 000 x g for 20 minutes. The precipitate was discarded and supematant was utilized for enzyme and inhibitory-enzyme assay. Enzyme and inhibitors, buffered with phosphate buffer, pH 6.9 containing 7 mM NaCI, were pre-incubated for 10 minutes at 370 C. Two per cent starch was utilized as substrate. After the addition of 10 ml of dinitrosalicylic acid (DNS), reaction was stopped at 1000 C and absorbance was measured at 530 DID. One a-amylase unit (lill) was defined as the amount of enzyme that would liberate 1 J.UIlol of maltose from the starch under the assay conditions (10 minutes, 37 DC, pH 6.9). The amylase inlubitor's activity (AlA) was calculated from the equation, through maltose generated: AlA = A 530 mn amylase - A 530 run plant extract A 530 mn amylase 247 1100 B.A.2.3 Lectins B.A.2.3.1 Collection of blood The rats were anaesthetized with ether and blood was immediately collected from abdominal aorta into centrifuged tube containing ADA (anticoagulant) [1 ml ADA: 5 ml blood]. The blood was then centrifuged for 15 minutes at 1200 rpm and then further centrifuged for 3 min. at 2200 rpm. Supematant was centrifuged after IS min. at 3200 rpm and the sediment was resuspended in 5 ml ofwashing buffer which was then centrifuged for 15 min. at 3000 rpm. The sediment was suspended in resuspcnding buffer [1 ml sediment: 20 ml resuspending buffer]. B.A.2.3.2 Estimation of lectins 109 of Amadumbe samples were homogenized with 200 ml of sodium borate buffer in shaker overnight. Samples were filtered with cheesecloth and serial dilutions were made as follows: 1:0, 1:1, 1:2, 1:4, 1:8, 1:10 and 1:20. Test tubes were prepared in duplicates and I ml of diluted extract, 0.5 ml of platelets obtained from rats were added together with thrombin. For the control, I ml of sodium borate buffer was added instead of the extract and for the blank only the buffer was used. Absorbance was measured at 546 nm at an interval of 0 and I minute. B.A.2.4 Total polyphenols [prussian Blue Assay - Price and Butler (1977)) 109 of each sample was homogenized in 100 ml of 2 M HCI and heated in a water bath for 95? C for 60 minutes. This was then cooled to room temperature (28? C ?2? C) and filtered. The filtrate was made up to 500 ml with distilled water. 1 ml of this extract sample was diluted with 50 ml of distilled water. Timed additions of3 ml of 0.10 M Fe~(S04)z were conducted and, exactly 20 minutes later, timed additions of 3 ml of 0.008 M K3Fe(CN)6 were started. Solutions were swirled and, after 20 minutes, absorbance was measured spectrophotometrically at 720 nm. A standard curve was prepared, expressing the result as gallic-acid equivalent. 248 B.A.2.4 Tannin [Van-Burden and Robinson (1981?) Weigh out 500 mg of the sample into a 50 ml plastic bottle. Add 50 ml of distilled water and shake for 1 hour in a mechanical shaker. Filter this into a 50 ml volumetric flask and made up to the mark. Pipette 5 ml of the filtered out into a test tube. Mix this with 0.008 M potassium ferrocyanide and 2 ml of 0.1 N HCL Measure the absorbance at 120 nm within 10 minutes. B.A.2.S Flavonoid [Bohm and Kocipai-Abyazan (1994?) 100 ml 80% aqueous methanol at room temperature was used to extract 10 g of the plant sample repeatedly. The entire solution was filtered through No 42 (125 mm) whatman filter paper. After transferring the filtrate into a cruCIble, it was evaporated into dryness over a waterbath. It was weighed to a constant weight. B.A.2.6 Saponin [Fenwick and OakeDfull (1981?) 20 g of sample material was weighed and placed in the Soxhlet extractor with acetone for 24 hours. Constituent parts, such as lipids and pigments, were thus removed. The solvent was changed to methanol and extraction was continued for another 24 hours. At this point, the method used so far (Hai et al., 1976) was modified. Instead of bringing the sample up to 250 ml as suggested in the original method, it was concentrated. The methanolic extract was then transferred to a rotary evaporator and evaporated to dryness. Dry extracts were suspended in approximately 10 ml of methanoL The colour was developed with 0.5 ml of 8 per cent vanillin solution in ethanol (freshly prepared for each determination) and 5 ml of 72 per cent (v/v) sulfuric acid was added to 0.5 ml of the methanol solution of the sample. The mixture was blended well, warmed in a water bath at 60? C for 10 minutes and then cooled in ice-cold water. The methanol blank with the reagents resulted in a strong yellow colour. Absorbance was recorded at 500 nm. Saponin was used as a standard to draw the standard curve. 249 B.A.2.7 CyanogeDic determination [O'Brien et aL (1991)] Amadumbe samples were homogenized with 160 ml of orthophosphoric/ethanol extraction medium for two minutes. The homogenate was washed on glass fibre filter. Total cyanogens: an aliquot of extract (0.1 ml) was added to 0.4 ml pH 7.0 buffer A (prepared from 0.1 M H~4 and 0.1 M Na3P04) in a stoppered Quickfit test tube, then j3-glucosidase preparation (0.1 ml), with the activity of 5 EU ml-1, was added. After a 15-minute incubation at 30" C, 02 M NaOH (0.6 ml) was added, followed by buffer A (2.8 ml; pH 6.0). Aliquots were assayed as in the colorimetric procedure descnbed below. Colorimetric procedure: Chloramine T reagent (0.2 ml: 0.5 per cent w/v) was added to 4 ml buffered extract in a stoppered Quickfit test tube and mixed well. Tubes were placed in ice/a water bath for 5 minutes, then pyridine-pyrazolone reagent (0.8 ml) was added in a fume cupboard. After 90 minutes, the absorbance at 260 nm was determined. The concentration of the samples was extrapolated from the standard curve, using KCN as a standard. Duplicate analyses were undertaken and blanks with the extraction medium were performed for each analysis. B.A.2.8 Oxalate [Munro and Bassir, (1969)] In this method, 2.0 g of each sample powder was extracted, with 0.15 per cent citric acid, for up to six hours. The solutions were filtered under vacuum. Prior to determination, the heavy metals in the acidified extracts were precipitated with 5 ml tungstophosphoric acid reagent and centrifuged at 1500 revolutions per minute for five minutes. The supematant was discarded and precipitates solubilized with hot, diluted H2S04. The content of each test tube was then titrated against 0.01 M KMn04. Titration with potassium permanganate can reveal the presence of oxalic acid. The acid is a weak reductant and needs an oxidant as strong as permanganate in order to react. Calcium oxalate was used as the standard and oxalate contents were expressed as an amount equivalent to 0.3 gllOO ml of calcium oxalate. 250 B.A.2.9 Alkaloid [Harborne (1973?) 5 g of the sample was weighed into a 250 ml beaker and 200 ml of 10 per cent acetic acid in ethanol was added, covered and allowed to stand for four hOUTS. The mixture was filtered and the extract was concentrated, using a water bath, to one-quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extract until precipitation was complete. The whole solution was allowed to settle and the precipitate was collected, washed with dilute ammonium hydroxide and then filtered. The alkaloid was the residue, which was dried and weighed. B.A.2.10 Phytate determination [Mehlich (1953?) 2 g of sample material was extracted in 30 ml of double acid (0.05 M HCI and 0.025 M HzS04) for three hours. Samples were filtered under vacuum through Whatrnan no 1 filter paper. The following solutious were added to 5 ml ofthe extract: ? 50 ml 0.05 M sulfuric acid; ? 20 ml ammonium molybdate; ? 20 ml ascorbic acid; ? 10 ml potassium antimonytartrase (the ratio 5:2:2:1). The mixture was allowed to stand for 30 minutes at room temperature to allow colour to develop. The absorbance of the samples was measured spectrophotometrically at 820 nm. A standard curve was prepared, expressing the results as a potassium hydrogen phosphate equivalent. The concentration ofphytate was calculated from its phosphorus content. B.Bl Isolation of amylase inhibitor B.Bl.l Extraction of Colocasia esc:ulenta a-amylase inhibitor from tubers Amadumbe tubers were obtained from the local market at Esikhawini in Kwazulu-Natal, South Africa. Tubers were washed, peeled, cut into small pieces (2 cm x 3 cm) and dried overoight at 40? C. The dried tubers were ground into flOUT, homogenized and defatted with hexane, then filtered and air-dried. 20 g of the defatted flour was extracted for a-amylase inhIbitor with 100 ml of distilled water containing I per cent polyvinylpolypyrrolidone (pVP). 251 The mixture was stirred continuously for two hours. The residue was re-extracted and the combined filtrate centrifuged at 12 000 g for 20 minutes. The precipitate was discarded and the crude extract supematant was subjected to 80% (NI4)zS04 saturation and left overnight at 4 DC. Precipitated protein was recovered after centrifugation (12 000 g x 20 min) and the protein pellet redissolved in a minimum volume of phosphate buffer (0.02 M, pH6.9, containing 0.3 M NaCl). The protein suspension was transferred to dialysis tubing and dialysed against the buffer (designated the annnonium sulphate extract). The dialysed sample was centrifuged and the fraction was analysed for amylase inlnbitor activity. B.B1.2 Ion-exchange chromatography The dialysed material, dissolved in 0.02 M phosphate buffer (pH6.9), was loaded on a column (6 x 1.1 cm) ofDEAE-Sephacel, equilibrated with the same buffer. The column was eluted with a gradient of (}-().5 M NaCl at a flow rate of 20 00 h-1. Four inhibitors, containing fractions eluted from the column, were pooled and analysed for protein and AI activity. The absorbance was measured at 280 nm and the graph plotted against tube numbers. B.Bl.3 Gel chromatography The ultimate purification procedure was performed by chromatography on Sephadex G-l00. During chromatography, the major peaks retained from ion-exchange dissolved in phosphate buffer were loaded on to a Sephadex G-IOO column (35 x I.I cm), equilibrated with the same buffer. The elution was performed at a flow rate of 15 00 h-I , fractions were pooled and analysed for protein and AI activity. Fractions (A-1 and B-2) with AI activities were collected, dialysed extensively, freeze-dried and dissolved in deionized water. B.Bl.4 Molecular weight determination by gel filtration chromatography The relative molecular weight (M,) of the native enzyme was determined by using a Sephadex G-100 column. Elution was carried out at the flow rate of 15 00 h-I , with an elution buffer comprising 50 mM tris-HCI pH 7.5. The calIbration curve was constructed using protein markers: 2 mg 00-1 cytochrome c (12 400), 3 mg 00-1 carbonic anhydrase (29 000), 5 mg 00-1 alcohol dehydrogenase (150 000) and 4 mg 00-1 13-amylase (200000). 2 mg ml-J of 252 Dextran blue was used to determine the void volume (Vo). A calibration curve between log log molecular weights of protein marlrers and the partition coefficient values (KAV) was constructed. B.BI.S Enzyme assay The a-amylase and a-amylase inlnllitor activity were measured using Bernfeld's method (Bernfeld, 1955). a-Amylase inhibitor extracts were added to a one-per-eent (w/v) starch solution in a 20 mM phosphate buffer, containing 0.4 mM NaCl (pH 6.9) and a-amylase. The mixtures were incubated at 37? C for 30 minutes. Reaction was stopped by the addition of 10 00 of dinitrosalicylic acid reagent (DNS). This solution was boiled for five minutes, then cooled and the absorbance was read at 530 urn. One amylase unit is defined as the amount of enzyme that will liberate I !1mol of maltose from starch under the assay conditions (pH 6.9; 37? C; five minutes). Inhibitory activity is expressed as the percentage of inlnllited enzyme activity out of the total enzyme activity used in the assay. B.BI.2 Kinetic studies For all kinetic studies other than pH, both enzymes obtained after gel-filtration on Sephadex G-IOO were used. B.BI.2.1 pH The effect of pH was checked using the following buffers (0.1 M): sodium acetate (pH 5), sodium phosphate (pH 6-7), Tris-HCI (pH 8) and glycine-NaOH buffer (pH 9-10). The AI activity was assayed at different pH values (pH I - pH 10) for five minutes at 3r C and the remaining amylase activity was determined. B.BI.2.2 Temperature The optimum temperature for inhibition of the inlnllitor was determined by assaying the amylase activity at temperatures ranging from 20? C to lOO ?c at pH 6.9. Coustant amounts (loo) of AI were preincubated with phosphate buffer (pH 6.9) for five minutes at 2-100? C. Remaining amylase activity was determined. 253 B.Bt.2.3 Assay of a-amylase inhibitor activity The specificity of AI-At and AI-B2 was evaluated against amylases from different sources: human saliva, porcine pancreas, sweet potato, barley, Bacillus species, Aspergillus oryzae a-amylases, dissolved in phosphate buffer pH 6.9 For inhibitory assays, a-amylase inIu.bitors were pre-incubated with a-amylase enzymes in phosphate buffer for 30 minutes at3? ? C. B.B2 Isolation of saponin B.B2.t Saponin extraction and isolation Saponins were extracted from white Amadumbe tubers (Colocasia esculenta) obtained from the local market at Esikhawini, KZN, South Africa. One kilogram of the tubers were air dried, powered and extracted five times at room temperature with 95 per cent ethanol. The ethanolic extract was concentrated under vacuum on a rotary evaporator at 40? C. The resulting extract (15.53 g) was suspended in 10 ml of water and partitioned into a CH3CI H20 (1:1, v/v) mixture to furnish the 30 ml CH3CI-solub1e fraction and an aqueous layer. The aqueous layer was extracted with 30 ml n-butanol (n-BuOH) to give 40 ml n-butanol and H20-soluble fraction. B.B2.2 Thin-layer chromatography Aliquots of 10 ~ of the methanol extract were manually spotted on to Silica gel 60 F254 AI plates (20 x 20 cm), using a glass capillary. The solvent extracts (10 ~ per plate) were applied as separate spots to a liC plate about 2 cm from the edge (spotting line). After sample application, the plates were placed vertically into a solvent-vapor-saturated liC chamber. Plates were developed in system: chloroform/methanol/water (65:35:10 v/v) and developed for a distance of 12 cm, air-dried and sprayed with anisaldehyde, sulphuric acid, acetic acid (95:5:5). The liC plate was dried for between five and ten minutes in an oven at 100?. Spots were visualized by gentle heating with hot air (90? C) for ten minutes. Detection was also done with iodine crystals. 254 B.B2.3 Column chromatography n-Butanol extract was further separated by column chromatography using glass columns with sintered glass filters. Silica gel (Merck) was loaded as slurry prepared in the respective solvents, eluting with CH3CI and methanol (until a ratio of 50:50 was reached). Fractions were collected under gravitational flow. Fractions were pooled according to nc analysis. B.B2.4 HPLC-UV analysis Analysis was performed using a Sbirnadzu liquid chromatograph system (palo Alto, CA, USA), equipped with antosampler and Prominence Diode Array detector. A Teknokroma nucleosil lOO C18 column (5 ~ x 25 mm x 4 mm) was used. A binary gradient elution system, consisting ofwater (A) and 10 per cent acetonitrile (B), was utilized and separation was achieved using the following gradient program: 0-15 min, 90 per cent The flow-rate was 0.1 mlImin and the system operated at room temperature (23 ? 10 C). B.B2.5 Gas chromatography mass spectrometry Gas chromatography/electron ionization mass spectrometry (GC-MS) was performed on a Agilent 6890 GC system, including a HP 5973 mass spectrometer equipped with an electron impact (El) source on an HP 5 MS capillary column (30 m x 0.25 mm x 0.25 ~). GC parameters were as follows: injector split I :40, helium carrier gas flow rate 30 cm/min. Initial temperature was set at 500 C (hold 2 minutes) and the temperature ramp was 200 Clmin to 300 ?C (10 minutes hold). Spectra were obtained over m/z 35-550. Injection volume was I J.1l. System control and data evaluation were realized using the Enhanced ChemStation software package, Gl70lBA Rev. 01.00, incorporating the NlST 98 MS spectral library for sample identification. B.B2.6 NMR NMR experiments were done on a Broker AM-400 spectrometer (Broker, Rheinstetten, Germany) equipped with an HX inverse probe CH:500 MHz; C13:125 MHz; solvent: pyrimidine-d5). System control and data evaluation were realized with the XWlNNMR software package, version 2.6 (Broker, Rheinstetten, Germany). 255 B.C.l Esterification of beta-sitosterol 200 mg of beta-sitosterol was dissolved in 200 mg of oleic acid at 80? C. The solution was added to 80 m! of a three per cent solution of sodium caseinate at 60-80? C under strong stirring. B.C.2 Analyses of digestive enzyme activities B.C.2.1 Preparation of tissues The intestine of each rat was divided into two portions: the proximal segment (duodenum) representing the upper intestine, and the mid and distal segments Gejunum and ileum) representing the lower intestine. The rat intestine was free of food and rinsed in 0.9 per cent NaCL A fraction was prepared from the two parts of the intestine by homogenizing the parts in 00 M phosphate buffer. The homogenate was centrifuged at 10000 x g for 10 minutes and the supernatant was frozen until required for enzymatic assays. B.C.2.2 Imaccharidases [DahIqvist (1968)] The principle of this method is as follows: an intestinal homogenate is incubated with the appropriate disaccharide. The disaccharidase activity is then interrupted by boiling the solution and the glucose liberated is measured with a glucose-oxidase reagent. Five hundred microlitres of substrate at 56 mM was added to 200 J.L1 of homogenate (100 III for sucrase and maltase) and incubated for 60 minutes at 37? C (15 minutes for maltase). The reactions were terminated by incubating at 100? C for five minutes. The liberated glucose was measured, using a Glucose (GO) Assay kit. The absorbance of each tube was measured spectrophotometrically at 540 nID. The activity was calculated as follows: Ilg glucose x F Units/m! ofhomogenate = 180x60xN Key F = dilution factor of the homogenate (1.6 for the sucrase and maltase; 1.4 for the lactase); 256 180 = molecular weight of the glucose; 60 = incubation time in minutes; N = number ofmolecules of glucose hberated by hydrolysis in each sugar (n = 1 for sucrose and lactose, n = 2 for maltose) [Rodriguez-Castilla et al., 1996]. B.C.2.3 ATPase [Vaslirhelyi et aL (1986)] The Na+!K'"-ATPase activity of freeze-thawed samples was determined by measuring the release of inorganic phosphate (Pi) associated with the hydrolysis of ATP. The samples (250 )l1) were added to 4750 )l1 of Reagent 1 [final concentration per litre: 100 mM of NaCl., 20 mM of KCl., 2.5 mM of MgCh, 0.5 mM of ethylene glycol tetraacetic acid (EGTA), 50 mM oftris-HCI (pH 7.4),1 mM of ATP, 1 mM of phosphoenolpyruvate, 0.16 mM of nicotineamide adenine dinucleotide (NADH), 5 kU of pyruvate kinase, 12 kU of lactate dehydrogenase]. After 300 s, 65 J.L1 of 10 mM ouabain was added to inhIbit ouabain sensitive ATPase activity. The Na+JK+-ATPase is composed of the stoichiometric of two obligatory major polypeptides, the a-subunit (- 112 kDa) and the f3-subunit (- 45 kDa). The binding sites for ATP, cations and ouabain are localized in the a-subunit, which is responsible for the atalytic activity of the enzyme. The change in absorbance was monitored at 340 llID. The Na+JK+-ATPase activity was calculated from the difference in A340 . 257 AppendixC OLEIC ACID STANDARD 230 235 240 258 l:TOFMS;j 'ol AppendixD CERTIFICATE FROM ETInCS COMMITIEE University ofZululand Ethics Committee CIO Dr Brent Newrnan Department of Zoology University ofZululand Private Bag 1001 Kwadlangezwa 3886 27 July 2005 To whom it may concern ETInCS EVALUAnON OF RESEARCH PROJECT PROPOSAL Ibis letter serves to confirm that Ms Ronalda McEwan (Student No 056257), registered for a Doctoral Degree in the Department of Biochemistry and Microbiology at the University of Zululand, in accordance with appropriate rules submitted a research project proposal to the Ethics Committee of the University of Zululand. The research project will investigate the Antinutritional constituent ofColocasia esculenta (Amadumbe), a traditional food crop in Kwazulu-Natal. Based on the research protocol stipulated the above-said Ethics Committee could find no reason to reject the proposed research provided that relevant internationally accepted procedures pertinent to the maintenance and experimental treattnent of laboratory held rats are adhered to. The smdents Supervisor and Co-supervisor, Prof AR Opoku and Prof T Djarova respectively, have indicated that appropriate procedures will be followed in this regard. The candidate is however advised to ensure that these procedures are correctly implemented, and that all facilities are available for inspection by members of the Ethics Committee from time to time. Should these facilities and procedures be found to be deficient in terms of internationally acceptable standards, the Ethics Committee, on behalf of the University of Zululand, reserves the right to call for the termination of experiments, which then mayor may not be permitted to continue following rectification of relevant problems. The candidate is also advised to consult relevant Medical Research Council of South Africa rules and regulations with regard to the use of animals for experimental purposes, particularly Guidelines on Ethics for Medical Research: Use of Animals in Research and Training, ISBN 1-919809-53-8, 2004. A copy of this document will be made available in electronic format by the Chairman of the Ethics Committee should this be required. Yours sincerely Brent Newman (PhD) Chairman Ethics Committee University ofZululand 259 Appendix E - Posters presented at conferences ~ntlnutntlonal constltuent of CO!OCdS'd 25c//enrd' ..\madumbel a traolt:lonal et OP tood In K\o'\iazulu :'\;J.tJ.1 - ~ --- - - -- -- - ---- -- - ----~-~---- ------ -- --- -- ------~ ~._._.?_._-----~_._._. __._._._.._._._._._---~_._._._.--.-.------_._.-------', I ~ . i !i To ...............-.it~...-....~.........~ CN'_~u. ?$ !i~~ dila oI_' '1KaIn ~..,......_oI.. 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