Inhibitory Assay of Cheilocostus Speciosus Leaves Extract On Amylase

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The Costus specious is an important plant that is the source of many therapeutically efficient compounds possessing many traditional and pharmacological activities. Its ability to inhibit the activities of the digestive enzymes has been widely studied. The a-amylases are calciummetalloenzymes based on the fact that they are unable to function in the absence of calcium (Daisy and Ignacimuthu, 2008). Through the random locations along the starch, many studies have shown that a-amylase can break down the long chains of carbohydrates and consequently generate maltotriose and maltose from amylose or maltose, glucose and "limit dextrin" from amylopectin. Because of its ability to act and function anywhere in the substrate, a-amylase tends to act faster compared to the amylase.

The results of our study indicate that the inhibitory percentage of highest sample concentration at 0.64mg/ml is 1% and the lowest sample concentration at 0.16mg/ml is -68%. The inhibitory effects of the extract increased with the increase of the sample concentration. The chemical inhibitors decrease the enzyme activities, usually through blocking the substrate from entering the active sites of an enzyme (Mioko et al. 2001). This result corroborates with those of other previous studies that reported that the use of the crude extracts of the hexane, ethyl acetate, and methanol from the C. specious have known to have a significant ability in reducing the blood glucose level in STZ diabetic rats significantly (Hasenah, Houghton & Soumyanath, 2006). The hexane extracts are more effective in lowering the glucose level as well as normalizing the biochemical parameters in diabetic rats and being more potent as compared to the acetate and methanol extracts (Supuran, 2013).

Thermal stability graph

As indicated in the graph, the inhibitory percentage of highest sample temperature at 600C is -47% and the lowest sample concentration at 800C is -81%. Increases the inhibitory effects of the extract decreases, when the sample concentration. The activities of enzymes are strongly affected by the changes in temperature. Fundamentally, each enzyme functions bests at an absolute temperature. As indicated in the graph, the activities of the amylase decrease at the value above and below a given point due to denaturation. For the amylase, denaturation refers to the loss of the structure and therefore renders it inactive (Kalailingam et al. 2011). It is not surprising based on the fact that the significance of the tertiary structures of in amylases function and non-covalent forces in the determination of its shape. The results indicated in this graphs highly corroborate those of other studies. These optimal pH and temperature are within the ranges reported for a-amylase from some COS by several authors (Sales et al. 2012).

Ammonium sulphate fractionation of extract of Cheilocostus speciosus leaves on amylase

The Ammonium sulfate is widely used for salting out the proteins. In this process, the sulfate often takes up the water molecules around the protein exposing hydrophobic sites (Azarkan et al. 2003). This group renders the protein molecules stick together and some out as a solution. The first step to assess the inhibition effects of COS extract on the amylase was to fractionate the crude supernatant using ammonium sulfate. 40-45% fraction showed maximum amylase activity.

The findings of this study validate those of previous studies by other others who found that the solubility of the proteins relies on, among other things such as the salt concentration of the solution (Hasenah, Houghton & Soumyanath, 2006). At a low concentration, the salt causes the stability of various charged groups on the protein molecule, thereby attracting the protein into the solution and therefore enhances the stability (Mani et al. 2010). However, the increase in the salt concentration accompanies the attainment of the point of the maximum protein solubility. The increase in the sample concentration of the ammonium sulphate implies that there is less water available to dissolve the proteins.

Effects on Trypsin

Inhibitory assay of Cheilocostus speciosus leaves extract on Trypsin

The COS contains the naturally occurring chemical that acts as the trypsin inhibitors. Usually, the trypsin enzyme activities depend decisively on the defined conditions concerning chemical substrates or components, the temperature, the pH and the nature and the strengths of the ions. Our results indicated that the inhibitory percentage of highest sample concentration at 0.040mg/ml is 63% and the lowest sample concentration at 1.28mg/ml is -74%. This means that the inhibitory effects of the extract decrease with the increase in the sample concentration increases. Enzymes activities display highest activities at their respective optimum conditions (Kalailingam et al. 2011). In this way, any deviations from the optimum lead to the reduction of the activity depending on the degree of difference. However, moderate variation generates a smaller reduction.

Thermal stability of Cheilocostus speciosus leaves extract on trypsin

Thermal stability of COS as a trypsin inhibitor according to our thermal graph shows that the inhibitory percentage of highest sample temperature at 990C is 392% and the lowest sample concentration at RTM0C is -92%. This trend offers an implication that the sample concentration increases with the increase in the inhibitory effects of the extract from the COS. The COSs extract contains various chemicals such as the hexane, ethyl acetate, and methanol. Fundamentally, each enzyme functions bests at a certain temperature (Mani et al. 2010). Like most chemical reactions, this study offers the rate of the trypsin-catalyzed reaction increases with the increase in temperature. A ten degrees centigrade rise in the temperature increases the activities of most enzymes from 50 to 100 percent.

Ammonium sulphate fractionization of extract of Cheilocostus speciosus leaves extract on trypsin

Ammonium sulfate influences the solubility of proteins. Various studies have shown that the presence of a given concentration of salt affects the stability of different charged groups of the protein molecule. Because trypsin is a protein in nature, enzyme purification can be conducted by the same set of procedures as in our study as those for proteins (Makkar et al. 2007). Regarding our studys ammonium sulfate fractionization graph, the inhibitory percentage of highest sample ammonium sulfate fractionization at 0% is -47% and the lowest sample concentration at 15% is -83%. The trend indicates that the inhibitory effects of the extract decrease when the sample concentration increases. The graph shows the decreasing curve effect of the inhibition and the two small deviations can be seen at 15% and 0%. This trend is based on the fact that the increase in the sample concentration of the ammonium sulfate implies that there is less water available to dissolve the proteins.

Effects on Pepsin Enzymes

Inhibitory assay of Cheilocostus speciosus leaves extract on Pepsin

Pepsin is an enzyme that plays a crucial role in breaking down the protein into smaller peptides. It is generated in the stomach and therefore constitutes to one of the main digestive enzymes in the digestive systems of humans and many other animals. The extract from the Costus plant contains various chemicals such as the methanol, hexane and ethyl acetate usually in the crude form. In our study, the results and findings of our study indicated that the inhibitory percentage of highest sample concentration of the chemicals at 1.28mg/ml is -42% and the lowest sample concentration at 0.040mg/ml is -12%. This offers an implication that when the sample concentration increases the inhibitory effects of the extract decreases. It is also indicated in the graph inhibitory graph that the decreasing curve is as a result of the inhibition and that a smaller deviation occurs one small deviation can be seen at 0.064mg/ml. These results are based on the fact that the pepsin chemical inhibitors present in the COS extract increase the velocity of the reaction and then activates the effect which is concentration depended. The chemicals therefore increases the enzymatic paths of pepsin through offering the room for bind themselves to one reactant also known as a substrate. Through this process, the pepsin lower the activation energy of the reaction that they are catalysing, hence the speed of reaction is increased.

Thermal Stability

The activities of enzymes are strongly affected by the changes in temperature where each enzyme functions bests at a certain temperature. Pepsin activities are not exceptional and are greatly affected by the variations of thermal conditions. The results of our study indicated that the inhibitory percentage of highest sample temperature at 990C is 246% and the lowest sample concentration at 40C is 137%. This provides an implication that when the sample concentration increases the inhibitory effects of the extract decreases. Similarly, the graph offered a depiction that the decrease curve effect of the inhibition and the one small deviation can be seen at 370C. The results corroborates with those of previous studies which showed that plant COS extracts showed significant inhibitory activity on carbohydrate metabolizing enzymes to reduce the postprandial blood sugar level (Sage, Pritzl, and Bornstein, 1980). The presence of ethanol extracts in the extract caused significant inhibitory activities of the pepsin, hence reduction of the enzymatic actions (Sage, Pritzl, and Bornstein, 1980).

Ammonium sulphate fractionization of extract of Cheilocostus speciosus leaves extract on pepsin

The sulphate often takes up the water molecules around the protein exposing hydrophobic sites. This group renders the protein molecules stick together and some out as a solution. In our study as indicated in the ammonium sulphate fractionazation graph, the inhibitory percentage of highest sample ammonium sulphate fractionization at 45% is 4% and the lowest sample concentration at 0% is -63%. This offers an implication that when the sample concentration increases the inhibitory effects of the extract increases. This trend is based on the fact that the incubation of pepsin solution for some time causes a slight decrease in conductivity of the hydrochloric acid. The hydrolysis of the proteins present in the pepsin preparation with the ultimate freeing of the amino groups, which combines with the HCL (Azarkan et al. 2003). These results are further substantiated by the fact that graph shows the decreasing curve effect of the inhibition and the one small deviation can be seen at 15%.

References List

Azarkan, A.E. Moussaoui, D. van Wuytswinkel, G. Dehon, Y. LoozeFractionation and purification of the enzymes stored in the latex of Carica papaya J Chromatogr, 790 (2003), pp. 229-238

Daisy, E., and Ignacimuthu: (2008). Influence of Costus specious (koen.) Sm. Rhizome extracts on biochemical parameters in streptozotocin induced diabetic rats, Journal of health science, 54: 675681

Hasenah Ali., Houghton, P.j., & Soumyanath A. (2006). a-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. Journal of Ethnopharmacology. 107: 449-455

Kalailingam P. Sekar A. D. Samuel J. S. Gandhirajan P. Govindaraju Y. Kesavan M. The efficacy of Costusigneus rhizome on carbohydrate metabolic, hepatoproductive and antioxidative enzymes in steptozotocin-induced diabetic rats, Journal of Health Science, 57(1), 2011, 37-46.

Mani P. Kumar A. R. Bastin T. M. Jenifer S. Arumugam M. Arumugam M. Comparative evaluation of extracts of C. igneus (or C. pictus) for hypoglycemic...

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