| Objective:The inhibitory effect and mechanism of DMC?extractive from Cleistocalyx operculatus?on pancreatic lipase?PL?were investigated.To increase the inhibitory effect of DMC on PL,the aqueous solubility of DMC was improved with PAMAM.Then we investigated the binding interaction between DMC and bovine serum albumin?BSA?with and without solubilization.Finally,the UPLC fingerprint of Cleistocalyx operculatus.was established and analyzed.Methods:?1?Porcine pancreatic lipase was used with 4-nitrophenyl butyrate?as substrate?for the in vitro assay.The interaction between PL and DMC was investigated through evaluation of PL activity,changes of reaction kinetic parameters,conformation and thermodynamic parameters by fluorescence spectroscopy and UV absorption.?2?Third-generation poly?aminoamide??G3.0 PAMAM?dendrimer was synthesized via a divergent method with ethylenediamine as primary core and methyl acrylate.G3.0PAMAM was characteirzed through FT-IR.The solubilization effect of PAMAM on DMC was detected by UV spectroscopy,and the effect of DMC with PAMAM on PL inhibitory activity was investigated.?3?In close to human physiological environment?pH7.4 Tris-HCl buffer?,the binding interactions between DMC/DMC-PAMAM and BSA were studied through fluorescence,synchronous and three-dimensional fluorescence spectra techniques.?4?In this paper,twenty varieties of Cleistocalyx operculatus buds were collected from different regions and the quality was evaluated based on Ultra Performance Liquid Chromatography?UPLC?fingerprint coupled with chemometrics analysis.UPLC method was carried out on a Waters BEH C18?2.1 mm×100 mm,1.7?m?column by acetonitrile?A?-0.1%aceticacid solution as mobile phase at a flow rate of 0.3 mL·min-1,the column temperature was 30?,the detector was PDA and the detection wavelength was 240 nm.Results:?1?Pancreatic lipase inhibition assay showed that DMC exhibited competitive inhibitor against pancreatic lipase with IC50 value of 50.01±3.56?mol/L.Fluorescence spectra results showed that DMC quenched the fluorescence of pancreatic lipase mainly through dynamic quenching mode while increasing the DMC concentration.The synchronous fluorescence spectra and three-dimensional fluorescence spectroscopy indicated that pancreatic lipase microenvironment was not changed by DMC.The thermodynamic parameters?G<0 showed that the combination was a spontaneous reaction,?H<0,?S<0 showed that the main forces were hydrogen bond or van der Waals force.?2?The structure of G3.0 PAMAM was confirmed by FT-IR compared with literature.DMC solubility with G3.0 PAMAM is about 485.25?g/mL,and the drug-loading rate is83.09 mg DMC/g PAMAM.Seven days cumulative release of DMC-PAMAM reached85.19%.Pancreatic lipase inhibition assay showed that G3.0 PAMAM could enhanced the inhibitory effect of DMC on PL,and IC50 was 14.50±2.10?mol/L.Fluorescence spectra results showed that DMC-PAMAM quenched the fluorescence of pancreatic lipase mainly through dynamic quenching mode while increasing the DMC-PAMAM concentration.The synchronous fluorescence spectra and three-dimensional fluorescence spectroscopy indicated that pancreatic lipase microenvironment was not changed by DMC-PAMAM.The thermodynamic parameters?G<0 showed that the combination was a spontaneous reaction,?H<0,?S>0 showed that the main force was electrostatic interaction.?3?Fluorescence spectra results showed that DMC quenched the fluorescence of BSA mainly through static quenching mode while increasing the DMC concentration.Under298 K and 310 K,DMC and BSA binding constant were 3.31×105 L/mol and 3.44×105L/mol respectively;DMC-PAMAM and BSA binding constant were 1.29×105 L/mol and1.13×105 L/mol respectively;DMC and BSA binding sites were 0.6972 and 0.9972respectively;DMC-PAMAM and BSA binding sites were 0.9867 and 1.0234 respectively.The synchronous fluorescence spectra and three-dimensional fluorescence spectroscopy indicated that BSA microenvironment was not changed by DMC.The?G<0 indicated that BSA-DMC and BSA-DMC-PAMAM interaction was a spontaneous process.The thermodynamic parameters?H<0,?S<0 showed that the main forces were hydrophobic interactions.G3.0 PAMAM does not change the binding interaction between DMC and BSA.?4?The UPLC fingerprint of Cleistocalyx operculatus was obtained with 19 common peaks,and 5 common peaks were identified using standard,and No.2,3,4,16 and 19 peaks were identified as gallic acid,catechin hydrate,SW6,DMC and myrcene respectively.The similarity of 20 batches of standard decoction of Cleistocalyx operculatus were greater than 0.9.The samples were broadly divided into two categories by HCA and PCA.S3,S4,S8,S10,and S12 are grouped into one category,and the rest are grouped into one category.DMC and Catechin may play an important role in quality control and evaluation of Cleistocalyx operculatus.Conclusion:?1?DMC had inhibitory effect on pancreatic lipas,and DMC was a competitive inhibitor of pancreatic lipase.DMC quenched the fluorescence of pancreatic lipase mainly through dynamic quenching mode.DMC has high affinity for PL and does not chang PL microenvironment.?2?G3.0 PAMAM can increase solubility and slow drug release of DMC.G3.0 PAMAM can enhanced the inhibitory effect of DMC on PL.Results show that PAMAM is a class of DMC solubilizers worthy of further study.?3?DMC and DMC-PAMAM quenched the fluorescence of BSA mainly through static quenching mode,during which hydrophobic interaction played dominant roles.DMC has high affinity for BSA.BSA microenvironment was not changed by DMC or DMC-PAMMA,and it implies that DMC may be low toxic.G3.0 PAMAM does not change the binding interaction between DMC and BSA.?4?The UPLC fingerprint of Cleistocalyx operculatus was established,and the method is precision,repeatability and stablility,simple and reliable,which shows that it can be used for quality control and evaluation of Cleistocalyx operculatus. |
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