Screening for pancreatic lipase natural modulators by capillary electrophoresis hyphenated to spectrophotometric and conductometric dual detection
2021
The search for novel pancreatic lipase (PL) inhibitors has gained increasing attention in recent years. For the first time, a dual detection capillary electrophoresis (CE)-based homogeneous enzymatic assay was developed employing both the offline and online reaction modes. The hydrolysis of 4-nitrophenyl butyrate (4-NPB) substrate catalyzed by PL into 4-nitrophenol (4-NP) and butyrate was monitored by spectrophotometric and conductimetric detection, respectively. The assays presented several advantages such as economy in consumption (few tens of nanoliters for online assays to few tens of microliters for offline assays), no modification of the target lipase, rapidity (< 10 min) and versatility. Tris/MOPS (10 mM, pH 6.6) was used as the background electrolyte as well as the incubation buffer for enzymatic reactions. The maximum velocity (Vmax) and the Michaelis-Menten constant (Km) of the PL catalyzed reaction obtained in the presence of dipalmitoylphosphatidylcholine (DPPC) vesicles added to mimic the lipid-water interface were in the same order of magnitude as those obtained in the absence of vesicles. Once validated, the method was used to screen crude aqueous plant extracts and purified compounds. We were able to identify the promising PL inhibition of hawthorn leaves herbal tea infusions at 1 mg mL-1 (37 ± 3 %) and PL activation by hawthorn flowers (22-26 %). Additionally, two triterpenoids purified from extracts of oakwood were identified for the first time as potent PL inhibitors demonstrating 51 and 57 % inhibition at 1 mg mL-1, respectively.
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