Studies On The Chemical Constituents And Bioactivities Of The Leaves Of Daphne Giraldii Nitsche

Daphne giraldii Nitsche, belonging to Daphne L of Thymelaeaceae, and widelydistributes in Shanxi, Shaanxi, Sichuan, Qinghai and Gansu provinces. The barks ofrhizomes and stems of D. giraldii, commonly called ‘Zu Shi Ma’ in Chinese folk medicines,has been used to treat aches and rheumatism. Pharmacological investigations showed thatZushima had anti-inflammation, analgesia, anti-fertility, anti-tumor, anti-thrombotic,anti-malarial, sedative and hypnotic activities.Zushima has long been used as a folk herb. In recent years, as the amount of Zushima tobe used in clinical practice increased, the herb resources became inadequate. It is necessaryto develop the leaves of Zushima into a new medical resources.The leaves of D. giraldii hasthe same kinds of chemical constituents as Zushima. In order to utilize this renewable part ofD. giraldii sufficiently, we studied the chemical constituents and pharmacological activitiesof the leaves of D. giraldii.Air-dried leaves of D. giraldii were cut into small pieces and then extracted with95%EtOH at reflux3times （2hours every time）, and the extracting solution combined and thenfiltered, filtrate was evaporated to remove EtOH. Then cool the solution to room temperatureand filtered, the sediment obtained was the part of the alcohol-soluble and water-insolublesubstance, and the filtrate was then subjected to solvent-solvent partitioning, to yieldpetroleum ether, CHCl3, EtOAc, n-butanol soluble phases. By using silica gel columnchromatography、TLC and recrystallizing methods,15compounds were obtained from theextracts. Structures were established and identified on the basis of chemical andspectroscopic evidence ((¹H NMR,¹³C NMR, HMBC). The structures of the15compoundsare as following: umbelliferone（1）, daphnetin-8-O-β-D-glu-coside（2）,7-hydroxy-8-methoxycoumarin（3）,8-hydroxy-7-methoxycoumarin（4）, daphnetin（5）, daphnoretin（6）,luteolin（7）,4′,7-dihydroxy-5-methoxyflavone（8）,5,7,3′-trihydroxy-4′-methoxyflavone（9）,β-sitosterol（10）, daucosterol（11）,4-hydroxy-benzaldehyde（12）, isosela-choceric acid（13）,pentadecane（14）, nonacosane（15）.Compound8and9are isolated from the D. giraldii for thefirst time.The effects of the extract of D. giraldii leaves on analgesia and inflammation were investigated on female ICR mice. The swelling auricles of mice induced by dimethybenzenewas used to observe the anti-inflammatory effect of the extract. Using the hot plate tests toobserve the effects of the extract on analgesia. The results of the experiments indicated thatthe extract inhibited the mice ear swelling in three dose groups（13mg/kg/day、26mg/kg/day、52mg/kg/day） remarkably（p<0.05，p<0.01）. And the extract decreased the heat stimulation ofmice in high dose groups（52mg/kg/day）. The heat pain threshold increased remarkably afteradministrated（p<0.05）, with the increased ratio being（23.7%²8.5%）. We concluded that theD. giraldii leaves has the effects on analgesia and anti-inflammation.To analyze and identify the constituents in mice serum after oral administration of theextract of D. giraldii leaves, UPLC-MS-MS was used as the analytic method. Theconstituents absorbed into blood were identified either by comparing the retention time andmass spectrometry data with that of reference compounds or by mass spectrometry analysis.Seven compounds absorbed into blood were detected, five of them were original in form,moreover, two metabolites were identified as glucuronide. The constituents absorbed intoblood and the possible metabolites which demonstrate to originate from D. giraldii leavesare responsible for the observed efficacy.The studies above indicated that the leaves of D. giraldii has the same kinds ofchemical constituents and pharmacological activities as Zushima. The serumpharmacochemistry of D. giraldii leaves should be subjected to complete investigation so asto illuminate the active mechanism of D. giraldii. The results of our studies providedevidence for the futher research on D. giraldii.