ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...
ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ... ANTI-NUTRITIONAL CONSTITUENT OF COLOCASIA ESCULENTA ...
B1.2.3.1 Extraction and purification ofa.-amylase inhibitor The scheme for the extraction and purification is shown in Figure B-1. Plant material IDefatted with hexane ICrude extract 11 %PVP(2x) 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 ofinfection 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 of0-0.5 M at the flow rate of 20 mIIh and 5 mI fractions were collected). The absorbance ofthe 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 ofamylase is defined as the amount ofenzyme 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 ofthe total enzyme 127
- 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 136 and 137: PURIFICATION AND AMYLASE INHIBITOR
- 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 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
- Page 187 and 188: Franco O.L., Rigden D.L., Melo F.R
- Page 190 and 191: Kokiladevi E., Manickam A and Thayu
- Page 192 and 193: Morton R.L., Schroeder RE., Bateman
B1.2.3.1 Extraction and purification ofa.-amylase inhibitor<br />
The scheme for the extraction and purification is shown in Figure B-1.<br />
Plant material<br />
IDefatted with hexane<br />
ICrude extract<br />
11 %PVP(2x)<br />
1Ammonium sulphate precipitation<br />
Extract<br />
1<br />
Ion-exchange chromatography<br />
1<br />
Gel chromatography<br />
Figure Bl-2.1: Extraction protocol for isolating a.-amykase inhibitor from<br />
Amadumbe tubers<br />
Tubers with no physical signs ofinfection were washed, peeled, cut into small pieces (2<br />
cm x 3 cm) and dried at 40° C for 24 hours. The dried material was milled (68 mesh)<br />
and the flour defatted with hexane and air-dried. Twenty grams of the defatted flour<br />
were added to 100 ml of distilled water (containing one per cent PVP), stirred for two<br />
hours and filtered. The residue was re-extracted and the combined filtrate centrifuged at<br />
12000 g for 20 minutes.<br />
126