A ‘Single Point’ Experimental Approach of Assessing the Inactivation Kinetics of β-glucuronidase by Aqueous-based Leaf Extractives Notably Used as Enemas

Excess beta-glucuronidase in the caecum and colon is responsible for enterohepatic circulation of toxic aglycones, which have been implicated in development of cancer, diarrhea, jaundice and other disease conditions of the gut. Botanicals are potential sources of molecules, which can serve as dietary components for inactivating beta-glucuronidase. Enzyme inactivation is employed in this study to categorize the potencies of the leaf extracts of Alstonia boonie (Ext1), Vernonia amygdalina (Ext2), Heliotropium indicum(Ext3) and Momordica charantia (Ext4) to inactivate beta-glucuronidase. In a ‘single point’ experimental approach a fixed amount of five micrograms of each samples were tested for their ability to reduce β-glucuronidase activity. Extracts reduced activity of the control (100%) to about 5-30% in 20 minutes. Ext2 and Ext3 were more potent as inactivators recording lower Km, Vmax and kcat compared to Ext1 and Ext4. Ext2 and Ext3 treatments resulted in 619 and 843 fold decreases in β-glucuronidase activity respectively. The partitioning ratios kinact/Km, kcat/kinact and Di values confirmed Ext2 and Ext3 as the more potent inactivators of β-glucuronidase than Ext1 and Ext4. Furthermore, Ext2/Ext3 inactivation mechanisms may involve the formation of a tenary enzyme-inactivator-substrate complex, which could be different from the inactivation mechanisms of Ext1/Ext4. However Ext2, Ext3 and Ext4 treatment produced same magnitude of order of the inactivation reaction, n = 0.17 and inactivation rate constant, kinact = 0.0024 min-1 compared with n = 0.09 and kinact = 0.0020 min-1 of Ext1 treatment which are indication of the complexity and form of the molecules in the extracts. Thus, extracts of similar chemical entities could display varying degrees of inactivation mechanism. These results show that the ‘single point’ experimental approach can be used to categorize the potencies of consumable botanicals for their ability to inactivate β-glucuronidase.