on-enzymatic glycation (glycosylation) is a multistage condensation reaction starting between reducing sugar and amino group (mainly in Lys and Arg) of different proteins (Stoynev et al., 2004) there are twofold meaning of non-enzymatic glycation: on one hand, early glycation product measurement which give estimation of glucose exposure and previous metabolic control of the subject; while on the other hand, intermediate and the late glycation reaction products measurement (Lapolla et al., 2005) ending up with complex heterocyclic compound formation called advanced glycation end products (AGEs) (Stoynev et al., 2004) lead in progression of atherosclerosis, Alzheimer's (Stoppa et al., 2006) and particularly in diabetes mellitus which is a endocrine disorder (Forbes et al., 2004) characterized by hyperglycemia and many chronic complications affecting the blood vessels, eyes, skin, nerves, and kidneys (Ahmad and Ahmed, 2006). Non-enzymatic glycosylation (Glycation) process, also known as Maillard reaction, (Hatfield, 2007) may contribute to formation of discoloration, off-flavors and decreased nutritional value (Nursten, 2005).
The intermediate appearance leads to the Amadori compound formation (an aldosylamine; aldose initial reaction with amino groups results in the formation of Schiff's base, which slowly rearrange itself for the production of 1-amino-1-deoxyketose, an aldosylamine) occurs in glycation early stages, however in late stage of glycation, irreversible formation of advanced glycation end products (AGEs) occur after a repeated reactions complex cascade as condensation, cyclization, dehydration, fragmentation and oxidation (Kikuchi et al., 2003). A state hyperglycemia found in diabetes, where non-enzymatic glycation, lipid oxidation and oxidation of protein occur. As a result, accumulation of advanced glycation end product (AGEs) in diabetic subject's tissues and the plasma. Accumulation of this AGE has been linked to pathogenic complication the development in diabetes (Lalla et al., 2001).
Research work was planned to find out the inhibition of glycation with natural inhibitor i.e. Sweet potato, turnip and methi.
To study the inhibitory effects on glycation or glycation inhibition invitro, eight combinations of each inhibitor were made with plasma and glucose, and were placed at 37ºC for five weeks (Zhang and Swaan, 1999). Plasma was used as a protein source. Samples were drawn after 1st, 2nd, 3rd, 4th and 5th week of incubation to perform the experiments for glycation and glycation inhibition. Along with temperature (37ºC) different concentrations of glucose and inhibitor were used.
Browning was estimated by taking absorbance at 370nm using spectrophotometer. After every week one sample was drawn and took 0.1 ml from it. Rest of the sample was kept in refrigerator at -20ºC. In 0.1 ml of sample 4ml of distilled water was added and 4.1 ml volume was obtained. Then absorbance was taken at 370nm by spectrophotometer. Blank samples will be run with each condition of glucose and inhibitor concentration. c) Total proteins estimation (g/dL)
Total proteins in all samples before and after dialysis were determined by Biuret method using Biuret reagent (Gornall et al., 1949). 1ml of Biuret reagent was added in blank, standard and all samples tubes. Placed the tubes at 37ºC for 15 minutes and reading was taken at 540nm. The standard curve was made with the half of absorbance of standard solution.
Glycated plasma samples were dialyzed against dist. H2O for twenty-four hours with constant stirring at room temperature to remove the free glucose by using dialyzing membrane.
The glycation level was measured by TBA method (Furth, 1988). b) Thiobarbituric acid (TBA) colorimetric technique TBA technique (Furth, 1988) was used for the determination of both enzymatic and non-enzymatic glycation. The standard curve was made by using fructose standard solution.
IV.
Combination of plasma with buffer and glucose showed maximum browning (0.233) at 1 st week of incubation while value of browning decreases to (0.196) at 2 nd week. In 3 rd week of incubation was at its minimum value (0.184). In the 4 th week it increases to (0.229) and in the 5 th week browning was (0.221). In the next combination of plasma with inhibitor sweet potato, glucose and buffer gives maximum level of browning. Plasma with buffer and glucose combination showed browning (0.168) at 1 st week of incubation while value of browning moves to maximum which was (0.177) at 2 nd week. In 3 rd week, incubation was at its minimum value (0.148). In the 4 th week it increases to (0.158) and in the 5 th week browning was (0.152). Combination of plasma with Turnip as inhibitor, glucose and buffer in the next showed maximum browning in the 1 st week of incubation which was (0.582) then it move to its lowest value of combination which was (0.307) in the 2 nd week. In the 3 rd week it gets (0.368) then in the 4 th week it was (0.353) and it shows 2 nd highest value of browning in the 5 th week which was (0.385). Combination of plasma with buffer and glucose showed maximum browning (0.286) at 1 st week of incubation while value of browning moves to minimum of its combination which was (0.253) at 2 nd week. In 3 rd week of incubation it starts increasing gradually which was (0.259). In the 4 th week it gets (0.265) and in the 5 th week of incubation browning was (0.276). In the next combination of plasma with Methi as inhibitor, glucose and buffer showed browning in the 1 st week of incubation which was (0.196) then it move to its maximum value of combination which was (0.225) in the 2 nd week. Combination of plasma with buffer and glucose showed browning (0.155) at 1 st week of incubation while value of browning increases to (0.161) at 2 nd week. In 3 rd week of incubation browning moves to maximum of combination which was (0.191). In the next combination of plasma with Turnip as inhibitor, glucose and buffer showed browning in the 1 st week of incubation (0.565) then the value of browning increases to (0.635) in the 2 nd week. In the 3 rd week it was lowest of combination (0.478) then in the 4 th week it showed highest browning of its combination (0.673) and value of browning in the 5 th week was (0.512). Incubation of plasma with glucose and buffer showed maximum glycation level at 1 st week of decreased glycation level (.280 mole/mole) recorded in 2 nd week. Combination of plasma, sweet potato as inhibitor, glucose and buffer showed highest value of glycation (.646 mole/mole) at 3 rd week of incubation which gradually decreases in coming two weeks. In case of glycation inhibition, inhibitor act as activator of glycation reaction as it showed minimum value (.394 mole/mole) in 1 st week of incubation. Incubation of plasma with glucose and buffer showed maximum glycation level at 4 th week of combination which was (.274 mole/mole) while decreased in glycation level (.169 mole/mole) recorded in 1 st week. Combination of plasma, turnip as inhibitor, glucose and buffer showed highest value of glycation (.908 mole/mole) at 3 rd week of incubation which decreases in coming week. In case of glycation inhibition, inhibitor act as activator of glycation reaction as it showed minimum value (.572 mole/mole) in 4 th week of incubation. Incubation of plasma with glucose and buffer showed maximum glycation level at 5 th week of combination which was (.342 mole/mole) while decreased glycation level (.274 mole/mole) recorded in 4 th week of incubation. Combination of plasma, methi as inhibitor, glucose and buffer showed highest value of glycation (.266 mole/mole) at 4 th week of incubation with a gradual increase from 1 st week.
V.
In case of non-enzymatic glycation, methanol extract of methi showed maximum inhibition of glycation in 3 rd week of incubation as compare to aqueous extract which showed minimum value of inhibition in 5 th week of incubation. On thorough study it is concluded that methanol extract of methi is more effective in glycation inhibition. Reference Références Referencias
1. Ahmad and Ahmed (2006) demonstrated that |
diabetes mellitus is a common endocrine disorder |
characterized by hyperglycemia and long-term |
complications affecting the eyes, nerves, blood |
vessels, skin, and kidneys. |
2. |
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