ABSTRACT
The aim of this study is to prepare a home grown anticoagulant which is cheap, readily available and yet provide the same biological effect as the anticoagulant in current use from African palm oil. The physiological anticoagulant is soured from animal, particularly porcine mucosa tissue called heparin. Heparin is a linear acidic sulphonated polysaccharide chain belonging to the family of glycosaminoglycan (GAG). The polysaccharide is made up of repeating disaccharide monomeric units of monosaccharide moiety, one of which is an iduronic acid (IdoA) and the other is sulphonated glucosamine (GlcNS). The IdoA can be acetylated at C5 to form glucuronic acid (∆UA). The GlcNS can sometimes be N-acetylated (GlcNAc). The reactive disaccharides are trisulphonated at C2 of IdoA and C2 and C3 of GlcN. Anticoagulants prevents blood from coagulation and is applied for the management of thromboembolic and cardiovascular related disease conditions. The preparation of the palm oil derived heparin (PODH) began with the alcohol precipitation of unsaponifiable fluid obtained from a mixture of 1:1 (v/v) palm oil: NaOH solution. The yield optimization was executed, using Box- Benhken experimental design (BBD). Design expert-11.3 version. Stability constant evaluation was carried- out using pH- metric technique of Bjerrum. The mathematical model of Calvin- Bjerrum was adopted for the calculation of Proton- GAG stability constant (𝑛̅𝐴 ). Metal – GAG formation numbers ((𝑛̅)) was calculated using the equation of Calvin-Rossotti. The negative logarithm of the Free- GAG Concentration (pL) was calculated using Iving-Rossotti mathematical model. The cumulative stability constant arising from the binding of GAG as a ligand to form binary complexes with divalent (M2+) and trivalent (M2+) metal ions such as Ca2+, Mg2+, Fe2+, Cu2+ and Zn2+ determined. The structural characterization of the test sample was investigated by the application of the complementary spectroscopy of UV- Vis, IR, ESI-MS, IH -NMR and 13C-NMR. Yield optimization of the process variables, 89.99 mls of palm oil : NaOH solution 1:1 (v/v ), for 12 weeks and 1:9 (v/v) of Ethanol increased the yield from 1.92 to 7.90%. Proton- GAG step – wise stability constant, revealed four (4H2+), associated with the isolated GAG molecule as a ligand, that is H4L compound as the isolated GAG. Iving- Williams order shows that the stability constant of the metal- ions studied is in the following trend: Zn2+ > Cu2+ > Fe3+ > Ma2+ > Ca2+. The UV-Vis analysis of the test sample and the reference sample showed maximum absorption at 203 and 247 nm. The bands showed chromophores of carboxylic and carbonyl groups arising from D-glucuronic acid and N-acetylated D-glucosamine of the xvi PODH, while weak absorption at 293 reveals GAG-associated molecules. A unique feature displayed by IR spectroscopy was the band at 594.00 nm for hypochloric acid. The other bands are normal features of sulphated heparin compounds. Mass spectroscopy revealed the differences between PODH and PMH. The fragmentation pattern confirms the presence of -OCl- at position 2, and uniquely reveals the absence of sulphonation at the C6 position. Three resonances peaks are normally displayed by 1H-NMR to indicate the presence of heparin. The reference sample showed the resonances of the three peaks at 3.90, 3.212 and 1.976 ppm whereas the PODH test sample showed 3.599, 3.287 and 1.85 ppm. Variation in source of the samples might be responsible for the chemical shift differences. The presence of aromatic preservatives also had a downward up field shift in the general chemical shift pattern of the test sample. It is noteworthy, that 3.519 ppm correspond to 6- hydroxy-sulphated glucosamine (GLcNS, 6OH), whereas, 3.90 ppm in the reference sample corresponds to 6-sulphated-, sulphated glucosamine (GLcNS6S), both 3.212 and 3.287 ppm corresponds to N-sulphated glucosamine (GLcNS.6x). Also, both resonance peaks at 1.976 and 1.850 for reference and test sample corresponds to the methyl signal (singlet) of N-acetylglucosamine (GLcAc) or CH3CO.The intensity of 1.14 obtained in this study for the test sample indicate better purity than the reference sample which gave 0.75 intensity levels.13C-NMR showed 12 cyclic carbons. The signals were observed at δ26 (cyclic carbon, C2); δ95 (CH-OH), a carbinol carbon while C4 and C1 carbons of the glycosidic bond had δ74. Carbonyl C existing in COOH (C6 ) and the cleaved amide group NH-CO-CH3 were observed at δ195. The signals for CH-OCl and CH-C-OCl were observed at δ31 and δ45 respectively at C2 and C3 . 1H-NMR and 13C-NMR spectrum were found to be in close agreement with the average units observed for the reference sample, additionally, The MS showed mass/charged ion of M/Z = 355.9 (M+ ). Matching the spectroscopic data indicated that the isolated compound (heparin) is a derivative of existing heparin of N-sulphonated D-glucosamine and N-acetyl-Dglucosamine polysaccharide. Thus, from the combined spectroscopic data and comparison with those described in the literature, the isolated compound is thus identified as a heparin derivative of repeating disaccharides of N-sulphonated-Dglucosamine and N-acetyl-D-glucosamine.
