Mode of pancreatic lipase inhibition activity in vitro by some flavonoids and non-flavonoid polyphenols
Introduction
An increase of triglycerides TG in blood is mainly due to intake of high lipid contained foods. Consequently, intestinal fat absorption of human is a very efficient process; approximately 95% of ingested fat is absorbed (Carey, Small, & Bliss, 1983) Digestive lipase in the small intestine is an essential enzyme that hydrolyzes lipids, the ester bonds in TG, to form fatty acids and glycerol. The inhibition of pancreatic lipase (PL) is, therefore, regarded one of the efficient ways to decrease blood TG in normal people. Moreover, inhibition of lipase results incomplete digestion of lipid and hence allows lipid to coat food particles and therefore interferes with the breakdown of other food components such as protein and carbohydrates and thereby exerting an anti-obesity activity.
In search of biologically active PL inhibitors from natural resources, various plant extracts and their phytochemicals have been screened for their lipase inhibitory activity. A growing number of literatures have suggested that polyphenols from vegetables, fruits, and cereals can inhibit some digestive enzymes in vitro (de la Garza et al., 2011, Gondoin et al., 2010, McDougall et al., 2009, Slanc et al., 2009). On the other hand, in vivo experiments with oligomeric procyanidins contained in apple polyphenol extract showed reduced triglyceride absorption by inhibiting PL activity in mice and humans (Sugiyama et al., 2007). The inhibition ability of these agricultural products is generally correlated to the phenolic content of their solvent extracts; for example, in our earlier study Vigna species of legume extracts with high polyphenol content showed PL inhibitory activity (Sreerama, Takahashi, & Yamaki, 2012). Other studies characterized the lipase inhibitory activity specifically correlated to their content of anthocyanins (Yao, Xu, Zhang, & Lu, 2013), proanthocyanidins (Gu et al., 2011, Nakai et al., 2005), flavan-3-ols (catechins) (Grove et al., 2012, Nakai et al., 2005), and phenolic acids (Cai et al., 2012). In fact polyphenols represent the major class for the PL inhibitors (reviewed by Lunagariya, Patel, Jagtap, & Bhutani, 2014).
Though a number of flavonoids and phenolic acids have been reported as inhibitory to the action of porcine PL in vitro, only a limited number of reports diagnosed their mode of inhibition, however, showing contradictory results (Grove et al., 2012, McDougall et al., 2009, Moreno et al., 2003, Nakai et al., 2005). To seek an experimental narrative on it, few selected flavonoid and nonflavonoid standards were analyzed for lipase inhibition potency and kinetics in vitro.
Section snippets
Chemicals
Lipase from porcine pancreas (Type II, 100–400 units/mg protein) was purchased from Sigma-Aldrich, USA. The lipase assay kit, Lipase Kit S (DS Pharma Biochemical Co., Osaka, Japan) contained 2, 3-dimercapto-1-propanol tributyrate (BALB) as substrate in sodium dodecyl sulfate (SDS) as solution, esterase inhibitor in phenylmethylsulfonyl fluoride (PMSF) solution, 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) as chromogenic reagent, reaction terminator, and buffer solution. The Catechins kit,
Results and discussion
Despite tremendous research to find out potential PL inhibitors, very few compounds have entered the clinical studies because of a lack of clear understanding of their structure–activity relationship (SAR). Most of the published researches on lipase inhibitors from food extracts mainly reported the inhibitive activity against PL by determining their IC50 values only. However, the determination of IC50 value for inhibitors is a primary type of analysis performed for SAR measurements for
Conclusion
Results of the present study using the most common microplate format used in academic research laboratories i.e., 96-well (8 by 12 matrix) with a typical reaction volume of 120 μL per well have demonstrated a competitive type of inhibition by three among all of the flavonoids and non-flavonoids tested. Interestingly, all of them, GA, EGC, and EGCG showed structural resemblances having a galloyl moiety in their structural backbone. It was further substantiated by another galloyl moiety containing
Acknowledgments
This research work was supported by Kirin Holdings Co., Ltd. Tokyo, Japan under UNU-Kirin Fellowship Programme during 2014 to 2015. KY conceived, designed, and supervised the study. ATMAR designed and conducted experiments, analyzed the data, and drafted the manuscript. YT facilitated the study.
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