ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...

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PURIFICATION AND AMYLASE INHIBITOR COLOCASIA ESCULENTA SUMMARY SECTIONB CHARACTERIZATION OF AND SAPONIN PRESENT 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 ofanimal 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 ofColocasia 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 a- IN
B1.1.1 Introduction CHAPTER Bl-l: a-AMYLASE INIDBITOR (AI) LITERATURE REVIEW 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 oftwo 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
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ANTI-NUTRITIONAL CONSTITUENT OF COL
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ABSTRACT Colocasia esculenta 1. Sch
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• My colleagues Kayode, Stranger,
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CHAPTER A-2 : MATERIALS AND METHODS
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CHAPTER 8-1-4 : DISCUSSION Bl-A.l I
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C.3.3 EXPERIMENTAL TEST C.3.3.1 Foo
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MakP Makatini purple MakW Makatini
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Figures C3-9 Figure B-1 The activit
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Table C3-3 TableC3-4 Summary ofseru
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crop. Amadumbe is not extensively c
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section of the study therefore inve
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Figure AI-2: The aboveground portio
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Taro is produced in the Pacific Isl
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A.1.4 Anti-nutrients Foods are comp
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essential amino acids not available
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A.l.4.2 Amylase inhibitors In order
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A.l.4.4 Total polyphenols Polypheno
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A.1.4.7 when the levels are high en
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A.1.4.8 coordination sides to inhib
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However,ifsaponins have a triterpen
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A.l.6 nutritional factors naturally
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A.2.2.3 A.2.3 A.2.3.1 A.2.3.2 Speci
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A.2.3.4.2 A.2.3.4.3 A2.3.4.4 In ano
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A.2.3.6.3 \.2.3.6.3.1 \.2.3.6.3.2 L
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sulphuric acid. Absorbance was meas
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Table A3-1a: The moisture and ash c
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Table A3-1c Carbohydrate content (g
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and 4.13 mg 100 g-l DMin the unproc
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Table A3-3c: Fatty acids identified
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Table AJ-4b: The phenolic compounds
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The results of the analysis of alka
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MtW MtP EW EP MakW MakP Figure A3-3
Page 87 and 88: The crude fat analysis ofZululand C
Page 89 and 90: The relative proportion ofNa, Ca, K
Page 91 and 92: TIA indicated that the trypsin inhi
Page 93 and 94: samples in this study were also hig
Page 95 and 96: The cyanogen levels for Amadumbe tu
Page 97 and 98: CHAPTERA-5 CONCLUSION A.5.1 Nutriti
Page 99 and 100: In conclusion, Section A ofthis stu
Page 101 and 102: Alfaia S., Ribeiro G., Nobre A, Lui
Page 103 and 104: Bhattacharyya A, Rai S. and Babu CR
Page 105 and 106: Chavan U.D., Shahidi F. and Naczk M
Page 107 and 108: De la Cuadra C., Muzquiz M, Burbano
Page 109 and 110: Fatty acids. (2008) CyberlipidCente
Page 111 and 112: Grant G. (1991) Lectins. In: Toxic
Page 113 and 114: HofmanP., Vuthapanich S., Whiley A,
Page 115: Jobnson LT., Gee J.M, Price K.R., C
Page 118 and 119: MagaJA (1978) Simple phenols and ph
Page 120 and 121: Milton K. (2003) Micronutrient inta
Page 122 and 123: Noonan S.C. and Savage G.P. (1999)
Page 124 and 125: Pathirana C, Gibney MJ. and Taylor
Page 126 and 127: Quesada C., Bartolome B., Nieto 0.,
Page 128 and 129: Robertson RN., Highkin H.R., Smydzu
Page 130 and 131: Sheeba R. and Padmaja G. (1997) Mec
Page 132 and 133: Thompson L.U. (1988) Antinutrients
Page 134 and 135: Vijayakumari K, Siddhuraju P. and J
Page 138 and 139: calcium metalloenzymes, are complet
Page 140 and 141: Bl.1.2.4 Kunitz-Iike a-amylase inhi
Page 142 and 143: diabetes mellitus or obesity. Octiv
Page 144 and 145: B1.2.3.1 Extraction and purificatio
Page 147: BI.3.1 BI.3.2.1 Introduction CHAPTE
Page 152: B1.3.2.2 Kinetic studies The inhibi
Page 155 and 156: Bl.4.1 B1.4.2 Introduction CHAPTER
Page 157 and 158: Bl.4.3.3 results that amylase inhib
Page 159 and 160: The chemical structure ofits sapoge
Page 162 and 163: 12.1.2.3 Steroidal saponin The trit
Page 164 and 165: (the in vitro haemolytic activity)
Page 167 and 168: 82.2.3.2 82.2.3.2.1 B2.2.3.2.2 Air-
Page 169 and 170: 82.2.3.3.4 • ion source temperatu
Page 173: Figure 82-3.3: Chromatogram ofsapon
Page 180 and 181: Gamma- and possibly beta-sitosterol
Page 182: whereas beta-sitosterol was. It was
Page 185 and 186: Cheeke PR. (1971) Nutritional and p
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Franco O.L., Rigden D.L., Melo F.R
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Kokiladevi E., Manickam A and Thayu
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Morton R.L., Schroeder RE., Bateman
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Price K.R, Eagles J. and Fenwick GR
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Ukpabi U.R and Ukpabi J.U. (2003) P
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SECTIONC EFFECTS OF INGESTED BETA-S
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C.1.2.2 and fungal cells contain on
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Beta-sitosterol has been successful
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Digestive enzymes do the all import
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C.2.1 Introduction CHAPTERC-2 MATER
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C.2.2.3.3 C.2.2.4 C.2.2.4.1 C.2.2.4
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C.3.! Introduction CHAPTERC-3 RESUL
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C.3.3.3 Clinical signs The appearan
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C.3.3.5 ALT and AST values were sig
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.. - Figure C3-5: Representative ph
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Figure C3-7: Representative photomi
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C.4.1 Introduction CHAPTERC-4 DISCU
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significantly, decreased after beta
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decreased by sitosterols as a resul
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Bhattacharyya AI
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Dablqvist A (1968) Assay ofintestin
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Ivorra MD., D'OconMP., Paya M and V
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Miettinen TA, Tilvis RS. and Kesani
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Pollak O.I. and Kritchevsky D. (198
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2.1 Introduction CHAPTER 2 GENERAL
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2.4 Suggestions for future work Fur
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A.A.3 Enzyme trypsin 10 mg bovine-p
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AppendixB DETAILS OF MEmOOOLOGY BA1
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sugars were estimated from standard
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B.A.l.5.4.5 Liquid Chromatography M
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B.A.2.4 Tannin [Van-Burden and Robi
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The mixture was stirred continuousl
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B.C.l Esterification ofbeta-sitoste
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AppendixC OLEIC ACID STANDARD 230 2
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5 '11I;1 5 2 r - - - • j ,et [:=:
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