| Gentianella acuta (Michx.) Hulten belonging to the family of Gentianaceae is an annual plant. The whole herb has heat-clearing, damp-inhibiting, benefit bile and back the yellow efficacy. It is mainly used to treat hepatitis, jaundice, headache and fever in Mongolia native medicine. G. acuta contains various different components including xanthones, iridoids, flavonoids and lignans. Xanthones possess a lot of pharmacological and biological activities such as antiarrhythmic, antioxidant, protect ischemic cerebral damage, anti-inflammation and antibacterial activities.In this article the powder of dried G. acuta was extracted under reflux with ethanol-water (75:25, v/v), and then filtrated. The combined filtrate was then concentrated by rotary evaporator, and then the concentrated extract was suspended in water and successively treated with petroleum ether, methylene chloride, ethyl acetate and n-butanol. The n-butanol part was systematically isolated and purified by silica gel, ODS, Sephadex LH-20 column chromatography and HPLC preparation chromatography. As a result,9 compounds were separated and the chemical structures were fully determined as demethylbellidifolin(1), bellidifolin(2), swertianolin(3), norswertianolin (4), mangiferin(5), gentiopicroside(6), sweroside(7), oleanolic acid(8) and triptexanthoside C(9).We established a quantitative analysis of multi-components by single-maker(QAMS) method for simultaneous determination the content of the four active compounds including DMB, bellidifolin, swertianolin and norswertianolin from G. Acuta, DMB was chosen as the reference substance. We examined the liner range of the four components, the precision of chromatography system, the stability of test sample solution, the repeatability of the method, average recovery rate, the repeatability of RCFs and the chromatographic peak positional parameters by HPLC. The method was applied to determine the content of four xanthones from eight batches of G. Acuta, calculating the content of DMB, bellidifolin, swertianolin and norswertianolin by QAMS method and external standard method, respectively. Comparing the difference between the determination results of the two method. The results showed that all the RCFs have good repeatability, there was no significant difference between the two method.We developed, optimized and validated a simple, sensitive and specific UHPLC-ESI-MS/MS method for the simultaneous determination of the four active compounds in plasma samples. The method was applied to pharmacokinetics after oral administration of the G. acuta extract to rats. Butylparaben was employed as an internal standard. LC separation was achieved on an Agilent SB-C18 RRHD column (1.8μm,150 mm×2.1 mm) at 30℃ with an isocratic mobile phase consisting of acetonitrile-water (0.1% formic acid) (90:10, v/v). The detection was accomplished by MRM scanning with ESI source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for swertianolin, norswertianolin, bellidifolin, DMB and I.S. were 435.1/272.0,420.8/258.9,273.0/258.0, 258.9/214.9 and 193.0/92.0, respectively. All calibration curves of the four xanthones have good linear relationship in linear range, the LLOQ of the four xanthones are 4,82 ng/mL,5.11 ng/mL,2.90 ng/mL,3.21 ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 0.38±0.13 h for swertianolin,0.29±0.09 h for norswertianolin,1.08±0.19 h for bellidifolin and 0.96±0.09 h for DMB.We studied the chemical constituent of G. Acuta, and determined the contents of the four xanthones both in the plant and the rat plasma, then explored the pharmacokinetics of swertianolin, norswertianolin, bellidifolin, DMB for the first time. It is of great significance to clarify the chemical compositions and establish quality standard of G. Acuta; it is also important to develop new antiarrhythic drugs with good efficacy and own intellectual property rights. And our study providing important scientific basis for making full use of the rich natural plant resources of G. Acuta. |
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