Analysis Of Chemical Constituents In Naoxintong、Astragalus Membranaceus And Ardisia Gigantifolia Stapf

Objective To establish a method for the major content identification of Nao Xin Tong、Astragalus membranaceus、Ardisia gigantifolia stapf extract by using UPLC-Q-TOF-MS.Methods 1.Analysis conditions of NaoxintongThe columns were maintained at 30℃.The separation was carried out on an Agilent poroshell Acquity UPLC BEH C18(2.1 mm×50 mm,i.d.,1.7 mm),preceded by a C18 guard column(2.1×5.00 mm;Agilent,CA,USA).The mobile phase consisted of water containing 0.1%(v/v)formic acid(A)and acetonitrile(B).A gradient programwas used as follows:0-0.5 min,10%B;0.5-1.5 min,10-20%B;1.5-7.5 min,20%B;7.5-12.5 min,20-25%B;12.5-18 min,25-30%B;18-25 min,30-35%B;25-27.5 min,35-50%B;27.5-29,5 min,50-60%B;29.5-31.5 min,60-75%B;31.5-32.5 min,75-80%B;32.5-34 min,80-100%B;34-34.1 min,100-10%B;34.1-35 min,10%B.The flow rate was 0.2 mL min-1 and the sample injection volume was 2μL.Mass spectrometry was performed using an Agilent 6530 Q-TOF mass spectrometer(Agilent,Santa Clara,CA,USA)equipped with an electrospray ionization(ESI)interface and was operated in the negative ion mode with parameters set as follows:capillary voltage,3000 V;pressure of nebulizer,35 psi;drying gas temperature,300℃.The collision energy(CE)was set at 10-40 V and the mass range recorded was m/z 100-2000.2.Analysis conditions of Astragalus membranaceusThe columns were maintained at 30 ℃.The separation was carried out on an Agilent poroshell 120 EC-C18 column(2.7 mm×100 mm,i.d.,2.7 mm),The mobile phase consisted of water containing 0.1%(v/v)formic acid(A)and acetonitrile(B).A gradient programwas used as follows:0-10 min,5-50%B;10-20 min,50%B;20-22 min,50-90%B;22-25 min,90%B.The flow rate was 0.35 mL min-1 and the sample injection volume was 2μL.Mass spectrometry was performed using an Agilent 6530 Q-TOF mass spectrometer(Agilent,Santa Clara,CA,USA)equipped with an electrospray ionization(ESI)interface and was operated in the negative ion mode with parameters set as follows:capillary voltage,3500V;skimmer,500 V;pressure of nebulizer,35 psi;drying gas temperature,350℃.The collision energy(CE)was set at 45 V and the mass range recorded was m/z 100-1500.3..Analysis conditions of Ardisia gigantifolia stapfThe chromatography analytical procedures were performed on an Agilent 1260 Series(Agilent,Santa Clara,CA,USA)LC system.The columns were maintained at 30 ℃.The separation was carried out on an Agilent poroshell 120 EC-C18 column(2.1 mm×100 mm,i.d.,2.7 mm),preceded by a C18 guard column(4.00×2.00 mm;Agilent,CA,USA).The mobile phase consisted of water containing 0.1%(v/v)formic acid(A)and acetonitrile(B).A gradient programwas used as follows:0-10min,5-30%B;10-15 min,30%B;15-20 min,30-40%B;20-25 min,40%B;25-30 min,40-90%B.The composition was then held at 90%B for 5 min and returned to the initial conditions and maintained for 10 min for equilibration.The flow rate was 0.35 mL min-1 and the sample injection volume was 3μL.Mass spectrometry was performed using an Agilent 6530 Q-TOF mass spectrometer(Agilent,Santa Clara,CA,USA)equipped with an electrospray ionization(ESI)interface and was operatedin the negative ion mode with parameters set as follows:capillary voltage,3500 V;fragmentor,150 V;skimmer,65 VOCT 1 RF Vpp,750 V;pressure of nebulizer,35 psi;drying gas temperature,300 ℃;sheath gas temperature,350℃.The collision energy(CE)was set at 45 V and the mass range recorded was m/z 100-2000.Results Firstly,analysis of chemical constituents in Naoxintong capsules and their metabolites in vivo.UPLC/Q-TOF MSE is used to screen the chemical compositions in Naoxintong capsules.According to the precise molecular weights,fragmentation patterns and information from database,nineteen compounds were characterized in Naoxintong capsules.Such as(2S)-4’,5-dihydroxy-6,7-diglucose diflavanoiden、Safflomin A、Amygdalin、6-hydroxy kaempferol-3-RUTINOSE-6-Sauerstoff glucoside、6-hydroxy kaempferol--3,6-diglucose diflavanoiden、Paeoniflorin、CALYCOSIN-7-β-D-Glucoside、Kaempferol-3-O-β-rutinoside、5,7,4’-trihydroxy-6-methoxyflavone-3-O-β-D-Glucoside、Fisetin、CALYCOSIN、MORIN、lithospermic acid B、Formononetin-7-β-D-glucoside、CROCIN I、3’-hydroxy-4’-Methoxy、Genistein.Formononetin、Astragaloside V。Secondly,analysis of chemical constituents in Astragalus membranaceus.A HPLC-ESI-QTOF-MS/MS method is used to screen the chemical compositions in Astragalus membranaceus.According to the retention times,precise molecular weights and fragmentati CALYon patterns,a total of eighteen compounds were characterized from Astragalus membranaceus.Such as COSIN-7-β-D-Glucoside、CALYCOSIN、Formononetin-7-β-D-Glucoside、PRATENSEIN(P)-7-O-β-D-Glucoside、5’,7-dihydroxy-3’-Methoxy、3’-hydroxy-4’-Methoxy、PRATENSEIN(P)、Astragaloside Ⅶ、Astragaloside Ⅴ、Astragaloside Ⅳ、Agroastragaloside Ⅲ、Agroastragaloside Ⅱ、Agroastragaloside Ⅰ、Astragaloside Ⅱ-Sojabohnen、Isoastragaloside 11、Astragaloside Ⅰ、Acetyl astragalusThirdly,analysis of chemical constituents in Ardisia gigantifolia stapf.A HPLC-ESI-QTOF-MS/MS method is used to screen the chemical compositions in Ardisia gigantifolia stapf.According to the retention times,precise molecular weights and fragmentation patterns,a total of Twenty-seven compounds were characterized fro m Astragalus membranaceus.Such as bergenin、Ardisicrenoside D、Ardisicrenoside C、3-β-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-a-L-arabinopyranosyl-16a,28-dihydroxy-olean-12-en-30-oic acid-30-O-[3’-O-(α-D－glucopyranuronate)glycerol(1’→30)]ester、3-β-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyr anosyl-(1→2)]-a-L-arabinopyranosyl-16α,28-dihydroxy-olean-12-en-30-oic acid-30-O-[3’-O-(α-D-glucopyranuronate)glycerol(1’→ 30)]ester、3-β-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-a-L-arabinopyranosyl-16α,28-di hydroxy-olean-12-en-30-oic acid-30-O-[3’-O-(α-D-glucopyranuronate)glycerol(1’→30)]ester、3β-O-{β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-[β-D-glucopyrano syl]-(1→2)]-α-I.-arabinopyranosyl}-16-oxo-28-hydroxy-olean-12-en-30-oic acid 30-O-β－D-glucopyranosyl ester、Ardisianoside G、3β-O-β-D-glucopyranosyl-(1→2)-[β-D-xyl opyranosyl-(1→2)-3-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl-13β,28-epoxy-16α,20-dihydroxy-30-noroleanane、ardisicrenoside H、ardisicrenoside G、3β-O-{β-D--xylopy ranosyl-(1→2)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1→2)]-α-L.-arabinopyranosyl)}-13β,28-epoxy 16α,30-oleananediol、Ardisicrenoside A、3β-O-β-D-glucopyranosyl-(1→2)-[a-L-rhamnopyranosyl-(1 →2)-β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl-16α-hydroxyolean-30-al-28,13β-olide、lysikoianoside、3 β-O-β-D-glucop yranosyl-(1→2)-[a-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)]-a-L-arabinopyr anosyl-13β,28-epoxy-16a-hydroxyoleanane、3β-O-{β-D-xylopyranosyl-(1→2)-β-D-gluco pyranosyl-(1→4)-[β-D-glucopyranosyl]-(1→2)]-a-L-arabinopyranosyl}-13β,28-epoxy-16-oxo-olean-30-oic acid、cyclamiritin A-3β-O-{β-D-glucopyranosyl-(1 → 2)-β-D-gl ucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinopyranoside、cycl amiritin A-3β-O-{β-D-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinopyranoside、cyclamiritin A-3β-O-{α-L-rhamnopyr anosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-a-L-arabinopyranoside、3β-O-{β-D-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 →4)-[β-D-glucopyranosyl-(1 → 2)]-a-L-arabinopyranosyl)}-13β,28-epoxy-oleanan-16-oxo-30-al、16a-hydroxy-30,30-dimethoxy-3β-O-{β-D-xylopyranosyl-(1→2)-β-D-glucop yranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-a-L-arabinopyranosyl}-13β,28-epoxyolean ane、3-O-{α-L-rhamnopyranosyl-(1→2)-β-D-glu-copyranosyl-(1 4)-[β-D-glucopyrano syl-(1→2)]-α-L-arabinopyranosyl]-3β-hydroxy-13β,28-epoxy-oleanan-16-oxo-30-a1、3β-0-β-D-glucopyranosyl-(1→2)-[a-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl-13β,28-epoxy-16α-hydroxy-30,30-dimethoxyoleanane、3β-O-{β-D-xyl opyranosyl-(1→2)-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1→2)]-α-L-arab inopyranosyl)}-13β,28-epoxy-16-oxo-30-acetoxyoleane、cyclamiretin A-3β-O-α-_L-rhamn opyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranoside、primulanin.Conclusion This study introduces a comprehensive analysis method of chemical const ituents in Naoxintong、Astragalus membranaceus、Ardisia gigantifolia stapf extract.Th e results afford a significant guidance for the study of active constituents and quality control research for Naoxintong and Ardisia gigantifolia stapf.