Comprehensive analysis of hydrophilic fraction of crude drug extracts based on digestion and absorption processes

Proteins in foods are mostly degraded into amino acids during the protease digestion and absorption process in our body. Recently, it has been shown that some peptides and low molecular weight compounds in foods can resist protease digestion and can be absorbed into body to exert beneficial activities. To find candidate compounds which can be absorbed into blood circulation system, in vitro protease digestion is performed, especially in the field of food science. As well as proteins and peptides in food, those in crude drugs may be digested and metabolized in our body. However, few studies have focused on the hydrophilic fraction, containing peptides or low molecular weight compounds, of crude drug extracts. In this study, we applied a method to find candidate compounds, which are thought to resist protease digestion and can be absorbed in our body, from crude drugs.
In this study, six crudes drugs were examined. Methanol extracts of six crude drugs were dissolved in water, and fractionated into ethyl acetate soluble, n-butanol soluble, and water-soluble fractions. To perform in vivo protease digestion, water-soluble fraction was treated with proteases, including endo type proteinases and exo type peptidases. Amino group in the resultant digests were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent. AQC derivatives were subjected to a series of LC-MS/MS analyses. To screen the mass to charge ratio (m/z) of AQC derivatives, precursor ion scan, targeting product ion of AQC (m/z=171.0), was performed. To estimate the structure of compounds in peaks, product ion scan targeting the detected precursor ions was performed. To estimate the anti-inflammatory effect of the compounds, suppression of nitric oxide (NO) production in primary cultured rat hepatocytes was examined.
In this study, we identified isobutylamine, isoamylamine, and 2-methylbutylamine in the exopeptidase digest of hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder. In rat hepatocytes treated with interleukin-1β, all these decarboxylated amino acids suppressed the production of NO. These facts suggest that decarboxylated amino acids may be found in vivo after the oral administration of crude drugs and may be responsible for the anti-inflammatory effect of them.