Phytochemical profiling by UHPLC-DAD/±HESI-MS/MS analyzes and hepatoprotective activity of Gentiana cruciata L. against CCl4 induced liver injury in Wistar rats

Abstract

Many Gentiono species are known for their pharmaceutical values, such as Gentiono crucioto L, commonly called cross gentian [1]. The dried roots and above-ground parts of G. crucioto are consumed in the Balkan region as herbal tea or a medicinal wine for loss of appetite, as a stomachic and component in preparations showing beneficial effects in gall and liver diseases [2]. This study using in vivo model investigates hepatoprotective activity of G. crucioto aerial part methanol extract (GCA) against carbon tetrachloride-induced liver injury in rats. Wistar rats were orally pretreated with GCA (100, 200, and 400 mg/kg) and silymarin (100 mg/kg) for seven days before they were treated with CCl4 (1 ml/kg, 1:1 mixture in olive oil) which caused liver injury. Separation, determination and quantification of components in GCA was perform­ed using Dionex Ultimate 3000 UHPLC system equipped with a diode array detector (DAD) and connected to a triple-quadrupole mass spectrometer. Pretreatment with GCA dose-dependent­ly and significantly (p < 0.001) decreased levels of serum transaminases, alkaline phosphatase and total bilirubin, whereas an increase was found in the level of total protein compared with CCl4-treated group. In the liver tissue antioxidant studies, we found a significant increase in the levels of catalase, superoxide dismutase and reduced glutathione, whereas there was marked reduction in the levels of thiobarbituric acid-reactive substances, as compared to CCl4 treated group. Histological analyses also show that GCA reduced the incidence of liver lesions including necrosis, ballooning degeneration and micro- and macro-vesicular changes induced by CCl4 in rats. GCA was characterized by the presence of sweroside, swertiamarin, gentiopicrin, loganic acid, isovitexin 4',7-diglucoside, orientin and vitexin, as revealed by UHPLC-DAD-MS and UHPLC-MS/MS analyses. Quantification of targeted compounds in the SRM (selected reaction monitoring) experiment of UHPLC-MS/MS analysis clearly indicated that gentiopicrin (1.067%) was the dominant secoiridoid glycoside in GCA, whereas concentrations of sweroside (0.064%) and swertiamarin (0.033%) were significantly lower.