ABSTRACT
Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5-35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 μM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.
ABSTRACT
In this study, the chemical profiling of Jingyin Granules and the tissue distribution of nine major constituents in this Chinese medicine were performed after oral administration of Jingyin Granules to rats, by using UHPLC-Q-Exactive Orbitrap HR-MS. An Acquity UPLC BEH C_(18) chromatographic column(2.1 mm×100 mm, 1.7 μm) was used as solid phase, while the mobile phase was methanol and 0.1% formic acid water for gradient elution. The major constituents in this Chinese medicine were quickly and accurately identified, via comparison with the retention times and MS/MS spectra of the standards. A total of 106 chemicals were identified from Jingyin Granules, including 24 kinds of organic acids, 47 kinds of flavonoids, 10 kinds of iridoids, and 21 kinds of saponins and 4 kinds of other compounds. After oral administered Jingyin Granules to rats, 48, 30, 25, 23, 45, 34, 39, 26, 19 prototype compounds were identified in serum, heart, liver, spleen, lung, kidney, brain, fat, and testicles, respectively. Meanwhile, an LC-MS based analytical method was established for simultaneous determination of chlorogenic acid, swertiamarin, caffeic acid, sweroside, liquiritin, prim-O-glucosylcimifugin, arctiin, 5-O-methylvisammioside and arctigenin in biological samples. The tissue distribution(serum, liver and lung) of these nine aim constituents in rats after oral administration of Jingyin Granules were investigated. It was found that these nine constituents could be quickly absorbed into circulation system and then distributed to liver and lung tissues. Except arctigenin, the exposure of other eight aim constituents to serum and lung was peaked at 1 h. At 1 h, the exposure of these components to lung tissue were ranked as follows: swertiamarin [(75 191.0±3 483.21) ng·g~(-1)]>arctiin [(2 716.5±36.06) ng·g~(-1)]>5-O-methylvisammioside [(585.1±0.71) ng·g~(-1)]>arctigenin [(437.45±3.18) ng·g~(-1)]>chlorogenic acid [(308.1±5.66) ng·g~(-1)]>prim-O-glucosylcimifugin [(211.35±2.19) ng·g~(-1)]>sweroside [(184.3±9.05) ng·g~(-1)]>caffeic acid [(175.95±2.05) ng·g~(-1)]>liquiritin [(174.78±153.34) ng·g~(-1)]. In summary, an UHPLC-Q-Exactive Orbitrap HR-MS method has been established for rapid and accurate identification of the constituents in Jingyin Granules, while the tissue distribution of nine major absorpted constituents were investigated in rats following oral administration of Jingyin Granules. These findings provided key information and guidance for further studies on pharmacodynamic substances and clinical applications of Jingyin Granules.