The Chemical Constituent And Pharmacokinetic Studies Of Flavonoids And Triterpenoids On Astragalusmembranaceus (Fisch.) Bge. Leaves

Astragalus membranaceus is a Leguminosae Astragalus Linn plant,mainly produced in the Northeast and Northwest of China.As a kind of medicine,it is sweet and warm,return spleen and lung.It can tonifying Qi Yang,fixed perspiration,water andswelling,nourishing blood,and so on.Astragalus caused a great waste of medicinal resources by leaving its roots for medicine after picking.Modern studies have shown that the Astragalus membranaceus leaves have the pharmacological effects of regulating immunity,delaying senescence,protecting cardio-cerebrovascular vessels and antibacterial and anti-tumor.Therefore,for the protection of wild astragalus and the development of new medicinal resources,the active components and pharmacokinetics of flavonoids and triterpenoids of A.membranaceus leaves were studied as follows:In this article,A total of 15 compounds were isolated from ethyl acetate and n-butanol layer of A.membranaceus leaves by silica gel,ODS and HPLC preparation chromatography.As a result,11 compounds were identified by NMR and ESI-MS technique,including five flavonoids:kaempferol-3-O-[6"-O-(trans-p-coumaroyl)]-β-D-glucopyranoside(1);rhamnocitin-3O-β-D-glucopyranosyl-(1"-2")-β-D-apiofuranoslide(2);rhamnocitin-3-O-β-D-neohesperidos ide(3);rhamnoci tin-3-O-β-D-glucopyranoside(4);calycosin-7-O-β-D-glucoside(5),fivesapo nins:huangqiyenin K(6);astragaloside Ⅳ(7);cycloaraloside A(8);daucosterol(9);soyasapo genol B(10),a lignin:dehydrodiconiferyl alcohol-4-O-β-D-glucopy ranoside(11)。A RP-HPLC method was established for simultaneous determination of three flavonoids and one lignin in A.membranaceus leaves,rhamnocitin-3-O-β-D-neohesperidosi de;rhamnocitin-3-O-β-D-glucopyranosyl-(1’"-2")-β-D-apiofuranoslide;rhamnocitin-3-O-βD-glucopyranoside and dehydrodiconiferyl alcohol-4-O-β-D-glucopyranoside.This method use a Diamonsil-C 18(5μm,4.6 mm×250 mm)as chromatographic column;the flow rate was set at 1.0 mL/min;the sample size is 10 μL;column temperature is 25℃;detection wavelength is 266 nm;mobile phase:acetonitrile(A)-water(B)gradient elution(0-30 min,15%A→45%A).Four kinds of composition in the corresponding linear range were showing good linear relationship.The precision of RSD is 1.62%～1.89%;The sample recovery is 98.87%～102.21%and RSD is 2.46%～3.31%;The RSD of stability is 1.79%～2.18%.In this study,a simple,accurate and efficient UPLC-MS/MS method was established to determine rhamnocitin-3-O-β-D-neohesperidoside,rhamnocitin-3-O-β-D-glucopyranoside,huangqiyenin K and astragaloside Ⅳ pharmacokinetics in Beagle dogs.And compared the effects of enteric capsule and gelatine capsule on metabolism of 4 analytes in Beagle dogs.Plasma samples were treated by methanol-sodium carbonate precipitation and sarsasaponin as I.S.LC separation was achieved on an ACQUITY UPLC? BEH Amide column(1.7 μn,2.1 mm×100mm)with an gradient mobile phase consisting of 0.1%formic acid(A)-0.1%formic acid acetonitrile(B):0-3min(70%-85%B).The detection was accomplished by MRM scanning with ESI source operating in positive ionization mode.The optimized mass transition ion-pairs(m/z)monitored for rhamnocitin-3-O-β-neohesperidoside,rhamnocitin-3-O-β-D-glucopyranoside,huangqiyenin K,astragaloside Ⅳ and I.S.were 609.1/300.9、463.5/300.9、687.6/143.1、807.7/627.4 and 463.5/300.9,respectively.All calibration curves of the 4 analytes have good linear relationship in linear range,the LLOQ of 4 analytes are 0.675 ng/mL,0.650 ng/mL,0.775 ng/mL and 1.05 ng/mL,respectively.The Tmax about the gelatine capsule of 4 analytes is 1.28±0.21 h,1.22±0.19 h,2.32±0.37 h,2.14±0.24 h,respectively.The Tmax about the gelatine capsule of 4 analytes is 2.23 ± 0.29 h,2.34±0.46 h,3.18±0.33 h,3.23±0.51 h,respectively.The recovery,precision,stability of the samples met the requirements of biological sample analysis.It is of great significance to develop the new medicinal resource of A.membranaceus leaves for protecting wild A.membranaceus resources,promoting the sustainability and comprehensive development and utilization of A.membranaceus medicinal resources.This article study constituents and pharmacokinetics of flavonoids and triterpenoids in A.membranaceus leaves in order to provide reference for the establishment of quality standard and further clinical application of A.membranaceus leaves.