Characterization of active antiplatelet chemical compositions of edible Citrus limon through ultra-performance liquid chromatography single quadrupole mass spectrometry-based chemometrics.

Citrus limon L. (lemon, family: Rutaceae) is the third most popular edible fruit among the Citrus species. Our previous study has shown the significant antiplatelet activity of lemon extracts. The aim of the present study is to identify the features (retention time, m/z) associated with the antiplatelet activity of lemons by correlating a platelet aggregation assay with ultra-performance liquid chromatography single quadrupole mass spectrometry-based chemometrics analysis. The primary bioactivity-guided test results revealed that the butanol (BA) and ethyl acetate (EA) liquid-liquid extraction sections of the ethanol extract of lemons had significant inhibitory effects on platelet aggregation. Upon further separating the combined BA and EA sections with a silica column, four different active fractions were obtained, and their LC-MS data were collected. After modeling by two multivariate statistical techniques, namely, principal component analysis and orthogonal partial least squares discriminate analysis seven markers were predicted, identified, and tentatively classified as priority markers of bioactivity in lemons. Among them, the antiplatelet activity of four marker compounds, namely, oxypeucedanin hydrate, citric acid, diosmin, and limetin at concentrations lower than 300 μM was confirmed. Moreover, the specific mechanism of limetin interaction with the TP β receptor of thromboxane A2 and the effect of limetin on the PI3 K/Rap-1b signaling pathway through the βγ subunit of GPCR (i) in platelet aggregation were studied by differential proteomic analysis to illustrate the validity and persistence of these markers for application in lemon fruit platforms.