Antipsoriatic Proporties Of The Active Fraction From Flos Daturae And Their Pharmacological Mechanisms

Flos Daturae is the dry flower of Datura metel L. which belongs to the family of Solananeae. It has been recorded originally in 《Compendium of Materia Medica 》 and Chinese Pharmacopoeia》 (2005 version). Flos Daturae has bee used as traditional Chinese medicine for a long history and utilized widely to cure many diseases such as cough, asthma, convulsion and so on. Some investigation has demonstrated that the extracts from Flos Daturae have significant effect on psoriasis in clinic. The purpose of the present work was to: (1) further isolate and identify the active component from Flos Daturae;(2) evaluate and investigate the antipsoriatic profiles of the active component from Flos Daturae and their pharmacological mechanism.After a long process of experiment, finally isolate the active fraction from Flos Daturae for psoriasis with Sephadex LH-20 column chromatography and water-methol solvent system successly. Recesived flavonoids and withanolides constituents of the active fraction from Flos Daturae for psoriasis. More than 53% are flavonoids constituents.Used with silica gel, reversed-phase (ODS), Sephadex LH-20 column chromatography and HPLC, systemicly isolated flavonoids and withanolides constituents. According to UV, IR,¹H-NMR, ¹³C-NMR, ¹H-¹HCOSY,HSQC, HMBC and NOESY,1D,2D-NMR, combined with ESI-MS ,FAB-MS to identificate (Determination) the chemical structures. 10 compounds were isolated from flavonoids and withanolides constituents, Among them there are 2 flavonoids, 8 withanolides compounds. The structures of the 10 compounds were identified (Make certain) as: Kaempferol 3-O-β-D-gIucopyranosyl ( 1—>2 ) -P-D-glucopyranosyl -7-O-a-L-rhamnopyranoside;Kaempferol 3-O-a-L-rhamnopyra-nosyl (1—>6 ) -β-D-glucopyranosyl-7-O-β-D- glucopyranoside;Daturameteloside B (5α,27-dihydroxy- 6α,7α-epoxy- ( 20R,22R ) -l-oxo-witha-2,24 -dienolide -27-O-P-D-glucopyranoside);Daturameteloside C (5α,12p,27-trihydroxy-6α,7α-epo- xy-(20R,22R) -1- oxo-witha-2,24- dienolide- 27-O-β- D-glucopyranoside);Withametelin C (5α,6β,21-trihydroxy-l-oxo-witha-24-enolide);Daturameteloside F (5α,6β,7α,12β-tetrahydroxy-(20R,22R)-1 -oxo-witha-2,24-dienolide-27-O-P-D-glucopyranoside);Daturameteloside I (la,3p,12p,27-tetrahy- droxy- ( 20R,22R ) -witha-5,24-dienolide -3-O-(3-D-glucopyranoside);Daturameteline J (5a,6p,12p, 21,24,29-hexahydroxy-( 20R,22R ) -1-oxo- witha-2-enolide);Daturameteline K (5a,6p, 12(3,21, 24,25,29-heptahydroxy- ( 20R,22R ) -1-oxo- witha-2-enolide);Daturameteline L (5a,6p, 12p,27-tetrahydroxy-21,24-epoxy- (20S,22R,24R,25R) -1 -oxo-witha-2-enolide) ? Among 10 compounds that were isolated and identified from flavonoids and withanolides constituents, confirm 3 withanolides compounds as novel compounds, they are Daturameteline K, Daturameteline L, Daturameteline MeInder to investigate the kinds and number of the active fraction for the treatment of psoriasis from Flos Daturae, used 3 psoriasis models in vitro to check the effect of active fraction from Flos Daturae, withanolides and flavonoids constituents, Daturameteloside B and Daturameteloside C.On DMSO-induced injury in HaCaT cells MTT reduction and LDH leakage assays were used to observe the viability of HaCaT cells. 20-80 ug/mL of active fraction from Flos Daturae could dose-dependently decrease the injury of HaCaT cells induced by 4%DSM0. Furthermore, the flavonoids (5-20 ug/mL) , but not the withanolides constituents (30-240 Ug/mL) , could significantly relieve the injury induced by 4% DMSO in HaCaT cells. The findings suggest that the flavonoids of Flos Daturae may protect HaCaT cells from DMSO cytotoxicity assessed by MTT assay, which may be associated with improving mitochondrial function, but not membrane of HaCaT cells.On IFN-y-induced apoptosis in HaCaT cells MTT reduction and LDH leakage assays were used to observe the viability of HaCaT cells, assay of Hoechst 33342 staining and FCM to observe IFN-y-induced apoptosis, and Western blotting to check K 17 expression in HaCaT cells. 25-50 ug/mL active fraction from Flos Daturae could dose-dependently decrease the injury of HaCaT cells induced by IFN-y. Furthermore, the flavonoids (5-10 ug/mL) , but not withanolides constituents (21.5-86 ug/mL) , had significantly effect on IFN-y-induced apoptosis. Using Hoechst 33342 staining, compact granule fluorescence or fluorescence pieces in nuclecus and cytoplasm of apoptosis cell were found underfluorescence microscope, 25 ug/mL active fraction from Flos Daturae and 5 ug/mL flavonoids, but not withanolides constituents, could reduce fluorescence intensity of apoptosis pieces. In addition, the same result could be obtained with assay of FCM. The analyses of cell cycle found that active fraction from Flos Daturae, flavonoids and withanolides constituents did not alter cell cycle of apoptosis cell. Our data showed that 12.5-50 ug/mL active fraction from Flos Daturae, and 2.5-10 ug/mL flavonoids, but not withanolides constituents, could reduce the expression of K 17. The active fraction and flavonoids from Flos Daturae can protect HaCaT cells from apoptosis induced by IFN-y, which may be related to the inhibition of K 17 expression.On EGF-induced proliferation in HaCaT cells MTT reduction and LDH leakage assays were used to observe the viability of HaCaT cells. Using CLMS, the expression of K 17 and intracellular [Ca2+]i were observed. Withanolides constituents (43-86 |xg/mL), Daturameteloside B (3-30 ug/mL) and Daturameteloside C (3-30 ug/mL) could decrease the proliferation of HaCaT cells induced by EGF in dose-dependent manner, whereas active fraction from Flos Daturae (12.5-50 ug/mL) and flavonoids (2.5-10 ug/mL) had no effect on EGF-induced proliferation of HaCaT cells. The data indicated that EGF could up-regulate the expression of K17, whereas withanolides constituents, Daturameteloside B and Daturameteloside C, but not active fraction and flavonoids from Flos Daturae, could reverse over-expression of K 17. Furthermore, EGF could increase intracellular [Ca2+]i, whereas withanolides constituents, Daturameteloside B and Daturameteloside C could reverse a increase in intracellular [Ca2+]i of HaCaT cells. Withanolides constituents, Daturameteloside B and Daturameteloside C have inhibitory effect on EGF-induced proliferation in HaCaT cells, which may be related to inhibition of over-expression of K17 and intracellular [Ca2+]i. Taken together, flavonoids and withanolides constituents may be act as mainly component for the treatment of psoriasis in the active fraction from Flos Daturae. Our findings have demonstrated the protective effect of flavonoids on DMSO-induced injury and IFN-y- induced apoptosis in HaCaT cells, whereas withanolides constituents, Daturameteloside B, and Daturameteloside C have inhibitory effect on EGF-induced hyperproliferation in HaCaT cells. Both INF-y and EGF can produce the over-expression of K 17, and flavonoids and withanolides constituents can restrain the excessive expression ofK 17 in HaCaT cells of different states. The results indicate that the intervention of flavonoids and withanolides constituents in pathophysiological process of psoriasis may be mediated by different mechanisms. The important characteristics of the active fraction from Flos Daturae for psoriasis may be the integration of different pharmacological profiles to intervene the necroses or proliferation of skin cells in psoriasis, and it refelects the charactic of Chinese medicine that have multiple components, multiple levels, multiple targets actions. The molecular pharmacological mechanisms underlying the anti-psoriasis of the active fraction, active constituents and active component from Flos Daturae may be related to T cell activation, injury of inflammatory factor, increase in intracellular [Ca +]i and over-expression of K17.Flos Daturae;Psoriasis;Flavonoids;...