| ObjectivePhosphodiesterase?PDE?hydrolyzes phosphodiesters to phosphomonoesters,and is expressed in various tissues and organs intracellularly and extracellularly.The cyclic nucleotide phosphodiesters,both cAMP and cGMP,play important roles in cardiac physiology and pathology,can be hydrolyzed specifically by PDEs.In addition,seven of the 11 PDE family members are known to be expressed in the heart tissue,suggesting the importance of PDEs to the cardiac system.PDEs regulate many crucial processes maintaining cardiac function and other pathological signaling pathways.Encouraged by the success and broad applications of PDE inhibitors,investigations on the novel types of PDE inhibitors have drawn increasing attentions of researchers.Ilex pubescens Hook.et Arn.?Maodongqing in Chinese?is well-known for its roots,which is a traditional Chinese medicine used for the treatment of cardiovascular diseases such as coronary arterial thrombosis,heart failure,stroke and thromboangiitis obliterans for a long time.Our previous study has shown that Radix Ilicis Pubescentis extract dose-dependently improved the cardiac function and ventricular remodeling on rats with chronic heart failure.And our preliminary study additionally revealed that the extract from Radix Ilicis Pubescentis showed potent PDE inhibitory activities.However,the active compounds responsible for these in vitro and in vivo activities in the extract are still unclear.In this study,the chemical basis for the PDE inhibitory activity of I.pubescens roots was revealed for the first time using the established ultrafiltration LC-MS method.This work suggested that I.pubescens roots contained health beneficial compounds and can be potentially used as a functional source of medicine,soup and/or food for the improvement of cardiac diseases.Methods1.LC-MS separation and identification of the major constituents in I.pubescens roots.Roots of I.pubescens were pulverized into homogenized powder?No.80 mesh sieve?,which was extracted with methanol in an ultrasonic water bath.The extract was analysis with linear-gradient elution using mobile phases A?water containing 0.1%FA?and B?acetonitrile containing 0.1%FA?.The on-line UV spectra was recorded using diode-array detector and mass spectrums were recorded using electrospray ionization-ion-trap-time-of-flight-mass spectrometry.The MS fragmentations of compounds together with their retention times?tR?are summarized and compared with previous reports.Compounds were then temtatively characterized and further identified unambiguously by comparing their chromatographic and MS behaviors to corresponding standard compounds.2.Simultaneous determination of four chlorogenic acids and four Triterpene saponins in roots of 1.pubescens by HPLCRoots of I.pubescens were pulverized into homogenized powder?No.80 mesh sieve?,which was extracted with methanol in an ultrasonic water bath and then diluted with methanol to a concentration of 0.05 g·mL-1?in terms of raw material?.Standard compounds of chlorogenic acid,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,ilexsaponin B1,ilexsaponin B2,ilexsaponin A1 and ilexgenin A were accurately weighed and diluted with methanol into concentration of 12.5 u g · mL-1,29.6 ? · mL-1,30.0 ?g · mL-1,34.5 ?g·mL-1,169.5?g · mL-1,130.5?g · mL-1,324.0?g · mL-1 and 728.0 ?g · mL-1 respectively.All calibration solutions were prepared by serial dilution of the individual stock solutions of the standards with methanol and analyzed with the sample solution under the same condition using HPLC.Precision,repeatability,LOD and LOQ of the eight analytes were examed using the standard solution,along with the stability of the sample.Contents of the analytes in the samples were observed with the established method.3.LC-MS separation and identification of the active components with PDEI binding activities in I.pubescens roots.Sample solution in a concentration of 0.1 g · mL-1?in terms of raw material?was incubated with 20 U·mL-1 PDEI solution.The mixture was filtered by a centrifugal ultrafiltration device?Amicon???Ultra-4 10K,cut off molecular weight:10 kDa?.The ligand-enzyme complexes on the filter were washed with 50%methanol,which was collected and dried in nitrogen.The residue was dissolved in 200?L methanol and analyzed with liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry.The MS fragmentations of compounds together with their retention times?tR?are summarized and compared with previous data obtained from the I.pubescens roots extract.Compounds were then temtatively characterized and further identified unambiguously by comparing their chromatographic and MS behaviors to corresponding standard compounds.4.Relative binding efficiency with PDEI of the major constituents in I.pubescens roots.Sample solution in a concentration of 0.1 g · mL-1?in terms of raw material?was incubated with PDEI solution in concentrations of 0,10 U·mL-1,20 U·mL-1 respectively.The mixture was filtered by a centrifugal ultrafiltration device?Amicon???Ultra-4 10K,cut off molecular weight:10 kDa?.The ligand-enzyme complexes on the filter were washed with 50%methanol,which was collected and dried in nitrogen.The residue was dissolved in 200?L methanol and analyzed using HPLC.Standard compounds of isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,ilexsaponin B1,ilexsaponin B2,ilexsaponin A1 and ilexgenin A were accurately weighed and diluted with methanol into concentration of 10.24?g · mL-1,8.96?g · mL-1,7.84?g · mL-1,68.8?g · mL-1,53.6?g · mL-1 67.2?g · mL-1 and 475.2 ?g ·mL-1 respectively.All calibration solutions were prepared by serial dilution of the individual stock solutions of the standards with methanol and analyzed to obtain the calibration curves.Concentrations of the analytes in the samples were observed and calculated using the calibration curves.HPLC peak area enhanced as a result of incubation,which indicates binding activity of a ligand to PDEI.The enhancement factor was used for calculating the binding of a ligand to PDEI.5.PDEI inhibitory activities?IC50?of active compounds in I.pubescens roots in vitro.The analytes in the I.pubescens roots extracts in different concentrations were incubated with PDEI solution.The PDEI inhibitory assay was carried out spectrophotometrically using Cyclic Nucleotide Phosphodiesterase Assay Kit.OD value was recorded under 620 nm using an automatic microplate reader.Calibration curve of 5'-AMP were obtained for calculation of the release amount of 5'-AMP.The PDE inhibition activity was calculated compare to the control.The inhibitory activity was shown as the sample concentration needed to inhibit 50%of the enzymatic activity?IC50?and was calculated with the inhibition activities and the logarithmic concentration of the analytes using non-linear model in GraphPad Prism 5.6.PDE5A and PDE9A inhibitory activities?IC50?of active compounds in I.pubescens roots in vitro.The analytes in the I.pubescens roots extracts in different concentrations were incubated with PDE5A and PDE9A solution respectively.The PDE inhibitory assay was carried out spectrophotometrically using Cyclic Nucleotide Phosphodiesterase Assay Kit.OD value was recorded under 620 nm using an automatic microplate reader.Calibration curve of 5'-GMP were obtained for calculation of the release amount of 5'-GMP.The PDE inhibition activity was calculated compare to the control.The inhibitory activity was shown as the sample concentration needed to inhibit 50%of the enzymatic activity?IC50?and was calculated with the inhibition activities and the logarithmic concentration of the analytes using non-linear model in GraphPad Prism 5.Results1.LC-MS separation and identification of the major constituents in 1.pubescens roots.An HPLC method was established for simultaneous determination of major constituents in the roots of I.pubescens with optimization of the extraction condition for sample preparation.MS condition was optimized from the previous studies for characterization of major constituents in the roots of I.pubescens.Methanol extract of I.pubescens roots was analyzed in which eleven compounds were identified as chlorogenic acid,tortoside A,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,3,4,5-tricaffeoylquinic acid,ilexsaponin B3,ilexsaponin B2,ilexsaponin A1,ilexsaponin B1 and ilexgenin A respectively.2.Simultaneous determination of four chlorogenic acids and four Triterpene saponins in roots of I.pubescens by HPLCAn HPLC method was established for simultaneous determination of eight constituents of I.pubescens roots.Linear ranges of chlorogenic acid,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,ilexsaponin B2,ilexsaponin A1,ilexsaponin B1 and ilexgenin A were 1.25-12.5?g·mL-1,2.96-29.6?g·mL-1,3-30 ?g·mL-1,3.45-34.5?g·mL-1,13.05-130.5?g·mL-1,32.4-324 g·mL-1,16.95-169.5?g·mL-1,72.8-728?g·mL"1,respectively;the average recoveries?n=9?were above 95.0%and RSDs were less than 3.0%.Contents of the analytes in the samples were 0.13-0.15?0.23-0.27?0.31-0.37?0.27-0.28?1.24-1.65?2.42-2.86?2.18-2.81?8.29-10.44 mg·g-1,respectively.The methodology validation and test results indicated the established method to be efficient for content determination of chlorogenic acids and saponins in Radix Ilicis Pubescentis.3.LC-MS separation and identification of the active components with PDEI binding activities in I.pubescens roots.An ultrafiltration technique was used to concentrate and separate the active components with PDE I binding activities from the roots of I.pubescens.Then the active compounds was analyzed using the HPLC-DAD-IT-TOF-MS method established.The MS fragmentations of compounds together with their retention times?tR?are summarized and compared with previous data obtained from the I.pubescens roots extract.Eleven active compounds with PDE I binding activities in the extract of I.pubescens roots were identified as chlorogenic acid,tortoside A,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,3,4,5-tricaffeoylquinic acid,ilexsaponin B3,ilexsaponin B2,ilexsaponin A1,ilexsaponin B1 and ilexgenin A respectively.4.Relative binding efficiency with PDEI of the major constituents in I.pubescens roots.The enhancement factor was introduced to further understand the relative binding strength of the seven active compounds in order to screen the protential PDE I inhibitors.This factor is proportional to the LC peak area of the ligand bound to the target enzyme,thus the higher the factor means more bound compound.The seven active compounds in the order of binding activities were isochlorogenic acid A,ilexsaponin B2,isochlorogenic acid C,ilexgenin A,isochlorogenic acid B,ilexsaponin B,and ilexsaponin A1 accordingly.5.PDEI inhibitory activities?IC50?of active compounds in I.pubescens roots in vitro.The IC50S of isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,sidinafil,ilexsaponin B1,ilexsaponin B2,ilexsaponin A1 and ilexgenin A against PDE I were 779.5?M,1516?M,>106?M,44.79?M,459.5?M,313.1?M,158?M and 250?M respectively.6.PDE5A and PDE9A inhibitory activities?IC50?of active compounds in I.pubescens roots in vitro.The IC50s of isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C,sidinafil,ilexsaponin B1,ilexsaponin B2,ilexsaponin A1 and ilexgenin A against PDE5A were 193.5?M,436.8?M,>104?M,0.43?M,1801.7?M 48.8?M,22.4?M and 176.6 u M,respectively.The IC50s of the above compounds against PDE9A were>104?M,213.7?M,1452?M,2568?M,3944?M,8810?M,>105?M and 4546?M,respectively.ConclusionIn this study,an ultrafiltration LC-MS method was established for rapid screening potential PDE inhibitors for the first time.Eleven compounds from Radix Ilicis Pubescentis were identified as PDE-binding active compounds by this method.Further in vitro assays confirmed the PDE inhibitory activity of the seven components in Radix Ilicis Pubescentis.The strong inhibitory effects of ilexsaponin A1 and ilexsaponin B2 against PDE5A provide further understanding of the beneficial effects of I.pubescens roots on cardiovascular system,and also suggest that active products from natural plants/medicines can be health beneficial as edible and functional food sources.The success of this study suggested that the established approach could be a valuable tool for rapid screening of PDE inhibitors from I.pubescens and other complex samples. |
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