| Puerariae Flos that is botanically from the dried flowers of Pueraria lobata(Willd.)Ohwi and P.thomsonii Benth(Leguminosae)is the most representative traditional Chinese medicine used to alleviate hangover.Tectoridin is one of the major isoflavone found in flowers of P.thomsonii,which possesses pharmacological properties including hepatoprotective,hypoglycemic,anti-hyperlipidemic,estrogenic,anti-inflammatory and anti-oxidant,etc..In the present study,the chemical constituents of the flowers of P.thomsonii and the metabolism of tectoridin in rats were systematically investigated,which would provide scientific foundation for its clinical application,as well as searching for active metabolites for new drug development.The investigations were as follows.1.Thirty compounds were isolated from methanolic extracts of the flowers of P.thomsonii by means of solvent partition and column chromatographic methods,including Diaion HP-20,silica gel,ODS and preparative HPLC.On the basis of chemical evidence and spectral analysis,they were elucidated as 6"-O-acetyltectoridin(1),tectorigenin(2),tectoridin(3),tectorigenin-7-O-?-D-xylosyl-(1?6)-)-?-D-glucopyranoside(4),irisolidone-7-O-?-D-glucoside(5),calycosin-7-O-?-D-glucopyranoside(6),glycitein(7),glycitin(8),wistin(9),genistin(10),6"-O-acetylgenistin(11),6"-?--D-xylosegenistin(12),sissotrin(13),isotectorigenin-7-O-?-D-glucopyranoside(14),kakkasaponin ?(15),phaseoside ?(16),kaikasaponin ?(17),azukisaponin ?(18),kakkasaponin ?(19),baptisiasaponin ?(20),kaikasaponin ?(21),kaikasaponin ?(22),3-O-?-L-rhamnopyranosyl-(1?2)-?-D-galactopyranosyl-(1 2)-?-D-glucuronopyranosyl-abrisapogenol A(23),soyasaponin ?(24),soyasaponin ?(25),astragaloside ?(26),soyasaponin ?(27),Lathyrus saponin(28),luteolin(29)and vanillic acid(30).Among them,compound 1 was a new compound,compounds 6,11-12,14,18,20,22-23,25-26 and 28 were isolated from Pueraria genus for the first time,and compounds 7-9 and 13 were isolated from the flowers of P.thomsonii for the first time.2.An UHPLC/Q-TOF MS technology,as well as co-chromatographic analysis method was developed for the identification of tectoridin metabolites in vivo,by direct comparison of the retention time,UV spectrum with those of authentic sample or analysis fragmentation regularities of mass spectra.A total of 46 metabolites were identified in rat plasma,urine,bile and feces samples after orally given tectoridin(100 mg·kg-1),among them,24 metabolites were reported for the first time as the metabolites of tectoridin.Glucuronidation,demethylation,dehydroxylation,demethoxylation,sulfation,methylation,hydroxylation and reduction after deglycosylation at C-7 position were the major metabolic pathways of tectoridin in vivo.3.A HPLC-UV method was established for the first time to quantitative four metabolites tectorigenin-7-O-glucuronide-4'-O-sulfate(Te-7G-4'S),tectorigenin-7-O-glucuronide(Te-7G),tectorigenin-7-O-sulfate(Te-7S)and tectorigenin in rat plasma.Linear calibration curves of Te-7G,Te-7S and tectorigenin were obtained in the concentration range of 0.125-12.5,0.20-16 and 0.025-2.5 ?g·mL-1,respectively.The lower limits of quantitation(LLOQ)were 125,200 and 25 ng·mL-1,respectively.RSD was found to be less than 15%for both inter-day and intra-day precision assay.RE ranged from-10.5%to 14.5%for the accuracy assay.The extraction recovery of three analytes,as well as rutin were higher than 70.3%.Three metabolites shown stable when kept at-20? for 20 days,during three freeze/thaw cycles,at room temperature for 24 h,and at room temperature for 24 h after samples extracted,respectively.The pharmacokinetics parameters of four dominant metabolites were investigated in rats by the HPLC-UV method.The data obtained were fitted with a two-compartment open model and validated by DAS 2.1.1.The pharmacokinetic parameters for Te-7G-4'S,Te-7G,Te-7S and tectorigenin were summarized as follows:Tmax,3.50 ± 1.87,3.17 ± 1.81,5.58 ± 3.07 and 4.92 ±2.87 h;AUC(0-?),197 ± 79,198 ± 78,199 ± 91 and 98 ± 48 ?mol·h·mL-1.Furthermore,the mean area under the curve AUC(0-t)values for Te-7G-4'S,Te-7G and Te-7S comprised 30.3%,33.9%,and 22.6%of the total four tectoridin metabolites,respectively.And concentrations of Te-7G-4 S,Te-7G and tectorigenin shown two successive maximum concentrations,which occurred individually at 1.5 h and 8 h after treatment.In contrast,Te-7G did not appear during the same time period.4.An UHPLC/Q-TOF MS method was established for the first time to quantitative the metabolites in rat urine and bile after oral administration of tectoridin at different doses.Linear calibration curves of Te-7G-4'S,Te-7G Te-7S,tectorigenin,Ir-7G and irisolidone in rat urine were obtained in the concentration range of 0.20-100,0.10-100,0.08-40,0.02-25,0.04-20 and 5.0×10-3-2.0 ?g·mL-1,respectively.The LLOQ were 200,100,80,20.40 and 5 ng·mL-1,respectively.RSD was found to be less than 15%for both inter-day and intra-day precision assay.RE ranged from-13.1%to 14.7%for the accuracy assay.The extraction recovery of six analytes,as well as daidzein was higher than 85.2%.The matrix effects of four analytes ranged from 87.3%to 115.0%.Six analytes shown stable when kept at-20? for 20 days,during three freeze/thaw cycles,at room temperature for 24 h,and at room temperature for 24 h after samples extracted,respectively.Linear calibration curves of Te-7G-4'S,Te-7G,tectorigenin and Ir-7G in rat bile were obtained in the concentration range of 0.20-100.0.10-100,0.04-10 and 0.04-20 ?s·mL-1,respectively.The LLOQ were 200,100.40 and 40 ng·mL-1,respectively.RSD was found to be less than 15%for both inter-day and intra-day precision assay.RE ranged from-18.1%to 3.2%for the accuracy assay.The extraction recovery of four analytes,as well as daidzein was higher than 85.3%.The matrix effects of four analytes ranged from 85.9%to 119.1%.Four analytes shown stable when kept at-20? for 20 days,during three freeze/thaw cycles,at room temperature for 24 h,and at room temperature for 24 h after samples extracted,respectively.When tectoridin was orally administered to rats at the dose of 100 and 200 mg kg-1,16.9%and 15.4%of tectoridin was excreted as seventeen metabolites in urine within 72 h,among which,two major metabolites Te-7G and tectorigenin accounted for 5.5-5.5%and 4.3-4.4%.Furthermore,the cumulative excretion of tectorigenin and six glucuronided and sulfated metabolites in bile accounted for 7.8%and 4.2%of the dose within 60 h,among which,Te-7G and Te-7G-4'S accounted for 2.3-3.0%and 1.4-3.9%,respectively.The results indicate that the urine was the primary elimination route,and glucuronidation after deglycosylation at C-7 position was the major metabolic pathway of tectoridin in vivo.5.The inhibitory activity of tectoridin,kakkalide and their seven metabolites on rat lens aldose reductase were confirmed.Tectoridin and its five metabolites showed much stronger inhibition,and the potency(IC50,?M)of the inhibition was in order of Te-7S(1.36)>Te-7G(4.65)>tectorigenin(6.43)>tectoridin(12.1)>Te-7G-4'S(15.1)>Te-4'S(15.5),whereas kakkalide and its major metabolites Ir-7G and irisolidone showed no significant inhibition.The structure-activity relationships of the tectoridin and its metabolites on rat lens aldose reductase are elucidated as follows.The glycosylation of 7-hydroxy group tends to reduce the activity.Glucuronidation or sulfation at 7-hydroxy group increases the inhibitory activity.Sulfation or methylation at 4'-hydroxyl group appeared to reduce the inhibitory activity.Since tectoridin and its five major metabolites showed much stronger inhibitory activity on rat lens aldose reductase than kakkalide and its major metabolites,Ir-7G and irisolidone,the flower of P.thomsonii may be more effective than flower of P.lobata for diabetic therapy. |
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