picroside-i and kutkoside

Picrorhiza kurroa Royle ex Benth., is widely used in the Indian systems of medicine for the treatment of various liver ailments. Since, the role of oxidative stress in the pathogenesis of liver injury has become generally recognized, in present study the free radical scavenging effect of P. kurroa was assessed by on-line HPLC-DPPH and colorimetric DPPH methods. The comparative study on antioxidant activity of P. kurroa extracts by both methods revealed that colorimetric method showed very less free radical scavenging effect while HPLC-DPPH method showed high activity. Further, the kutkoside, an important ingredient of a potent hepatoprotective formulation "kutkin/picroliv" was investigated for its chemical composition by ultra-performance liquid chromatography coupled with diode array detection/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-QTOF-MS). Kutkoside was considered to be a single compound and reported as picroside-II or kutkoside, however, present investigation illustrated that kutkoside is a mixture of iridoid glycosides namely, picroside II, picroside IV and 6-ferulloylcatalpol.

Topics: Antioxidants; Biphenyl Compounds; Chromatography, High Pressure Liquid; Cinnamates; Dose-Response Relationship, Drug; Free Radical Scavengers; Free Radicals; Glucosides; Iridoid Glucosides; Picrates; Picrorhiza; Spectrometry, Mass, Electrospray Ionization

A rapid, sensitive and selective LC-MS-MS method for the simultaneous quantitation of picroside-I and kutkoside (active constituents of herbal hepatoprotectant picroliv) was developed and validated in rabbit plasma. The analytes and internal standard (Amarogentin) were extracted using Oasis HLB solid phase extraction cartridges. Analysis was performed on Spheri RP-18 column (10 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:MilliQ water (50:50, %v/v) as mobile phase at a flow rate of 1 ml/min with a retention time of 1.39, 1.33 and 1.42 min for picroside-I, kutkoside and amarogentin, respectively. The quantitation was carried out using an API-4000 LC-MS-MS with negative electro spray ionization in multiple reaction monitoring (MRM) mode. The precursor to product ion transitions for picroside-I, kutkoside and amarogentin were m/z 491 > 147, 199; 511 > 167, 235; 585 > 227, respectively. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (within- and between-assay variation), freeze-thaw (f-t), auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.5 ng/ml for both analytes. The recoveries from spiked control samples were > 60 and > 70% for picroside-I and kutkoside, respectively. Accuracy and precision of the validated method were within the acceptable limits of < 20% at low and < 15% at other concentrations. The analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of picroside-I and kutkoside post i.v. bolus administration of picroliv in rabbit.

Topics: Animals; Calibration; Chromatography, Liquid; Cinnamates; Glucosides; Iridoid Glucosides; Mass Spectrometry; Rabbits; Reference Standards; Reproducibility of Results; Sensitivity and Specificity