Isolation, Identification, Quantification And Pharmacokinetic Studies Of The Chemical Constituents Of Safflower

Safflower, the petals of Carthamus tinctorius, is one of the most important herbal drugs used in traditional medicine for promoting blood circulation and removing blood stasis. Flavonoids, fatty acid, polyacetylene and other types of constituents have been isolated and identified from safflower. However, there are no reports on the circulation levels of multiple safflower constituents after oral administration of safflower powder which is the main dosing form in traditional Mongolia medicine in which safflower has been used as the second most frequently used herbal medicine only next to the fruit of Terminalia chebula. Therefore, it is necessary to prepare pure safflower constituents and to establish a rapid, sensitive and selectivity method to simultaneously quantify the chemical constituents and to investigate their pharmacokinetic behaviors after oral administration of safflower powder.Various chromatographic methods were used to isolate and purify safflower constituents and the structures were determined by spectroscopic methods. A ultra-high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) with dynamic multiple reaction monitoring mode, which could extend the dwell time and produce a higher sensitivity and reproducibility comparing with conventional methods was established and used for the pharmacokinetic study of multiple safflower constituents.Twelve compounds were isolated and purified from safflower. The structures of these safflower constituents were determined as:hydroxysafflor yellow A, 6-hydroxykaempferol 3,6-diglucoside,6-hydroxykaempferol 3,6,7-triglucoside, 6-hydroxykaempferol 3-rutinoside-glucoside, kaempferol 3-O-glucoside, kaempferol 3-O-rutinoside, isoquercitin, chlorogenic acid, syringing,p-coumaric acid, protocatechuic acid, and 8Z-decaene-4,6-diyne-1-O-β-D-glucopyranoside (bidenoside C). Among the isolated safflower constituents, isoquercitin, chlorogenic acid and p-coumaric acid showed radical scavenging activity. The flavonoid aglycone 6-hydroxykaempferol obtained by hydrolysis of the major safflower flavonoid glycosides showed much stronger DPPH scavenging activity than the glycosides, suggesting that 6-hydroxykaempferol and other flavonoid aglycones play important roles for the in vivo bioactivity of safflower, as glycosides are known to metabolize to their aglycones in human. A rapid and validated UPLC-MS/MS method with dynamic multiple reaction monitoring mode was developed for the simultaneous determination of multiple safflower constituents. As a result, this method have successfully quantified the contents of the 11 constituents in safflower and been efficiently applied to the pharmacokinetic study of 5 compounds, hydroxysafflor yellow A, 6-hydroxykaempferol-3,6-diglucoside, chlorogenic acid,p-coumaric acid and bidenoside C in the rat plasma. Of these constituents, bidenoside C displayed the largest AUC0-t value followed by hydroxysafflor yellow A and other compounds, indicating the important roles of these compounds in the in vivo pharmaceutical effects of safflower. The favorable pharmacokinetic behavior of bidenoside C rendered the importance for further study of its bioactivity.