| The compatibility of traditional Chinese medicines(TCMs)is the characteristic of clinical medication of traditional Chinese medicine(TCM).Through the compatibility,through compatibility,there is interaction between TCMs,the purpose of increasing efficiency and reducing toxicity can be achieved.The traditional way of taking TCMs is decoction.TCMS decoction is recognized as a complex system which is composed of a variety of chemical components.It is a heterogeneous system formed by the mixture of true solution,colloid,suspension and emulsion.The traditional research on TCMs mostly focuses on small molecular compounds,while the high content of macromolecular compounds such as protein and the interaction between compounds are ignored.Studies have reported that in the process of TCMS decoction,the components can form supramolecular structure through self-assembly,and the pharmacodynamic effect also changes,which may be one of the mechanisms of enhancing efficacy and reducing toxicity of TCMs compatibility.Shaoyao Gancao Decoction(SGD)originates from "Treatise on Febrile and Miscellaneous Diseases" and is one of the classic prescriptions.In the prescription,Paeonia lactiflora Pall.(SY)and Glycyrrhiza uralensis Fisch.(GC)are mostly mixed in 1:1.The combination of the two drugs has antispasmodic and analgesic effects and is often used in the treatment of spastic diseases,painful diseases,inflammatory diseases and bronchial asthma.The current research on SGD mostly focuses on small molecular components,while the research on macromolecular components such as polysaccharides and proteins and the interaction between drugs is rarely reported.Therefore,it is necessary to deeply study the macromolecular components such as protein,and explore the interaction between the components in SGD based on molecular self-assembly,which can clarify the molecular mechanism and scientific connotation of the enhanced efficacy of SGD after compatibility,and provide new ideas and methods for exploring pharmacodynamic substance basis of TCMs.In this study,SDG is taken as the research object,we puts forward that a large number of proteins and small molecular compounds in SGD form stable colloidal particles through self-assembly in the process of heating and decocting,which may be one of the active ingredients of SGD.Therefore,in this study,the small molecule and macromolecular components of SGD were analyzed and determined,and the self-assembly process of various components in the decoction was simulated.It was clear that the protein in the decoction mainly belongs to GC.The target protein(GP)was extracted and isolated from GC and the sequence of GP was analyzed.It was found that the GP and paeoniflorin(PF)could forme colloidal particles through self-assembly.The main contents and results of this study are as follows:1 Physical and chemical characterization of SGDThe decoction process of SGD was characterized,and it was found that during the decoction process,the pH of the decoction gradually decreased to about 5,the particle size and the absolute value of zeta potential gradually increased and then stabilized.Transmission electron microscopy(TEM)was used to observe the morphology of the decoction,showing that the particles were distributed irregularly in bulk,and the particles were easily adhered to each other or aggregated.The ultra-high performance liquid chromatography-tandem mass spectrometer(UPLC-MS/MS)was used to measure the concentration of 7 small molecular compounds.The results showed the content of PF was the highest.The protein content of SGD was 51.78 mg/mL determined by BCA method.The polysaccharide components were analyzed and determined by phenol-sulfuric acid method and ion chromatography electrochemical method.The results showed that the polysaccharide content was 175.11 mg/mL,of which the content of glucose was highest(91.51 mg/mL).Small molecular compounds,proteins and polysaccharides were extracted and separated from SGD by alcohol precipitation,extraction and anion exchange chromatography.The small molecules were combined with polysaccharides and proteins simulate the decoction environment for self-assembly,and the content changes of small molecular compounds before and after assembly were measured.The results showed that except for isoliquiritigenin,the content of small molecular compounds was significantly decreased(P<0.01),among which the content of PF decreased the most,while the assembly rate of protein and polysaccharide to small molecular compounds had no effect significant difference.The colloidal particles in SGD were prepared by differential centrifugation,and the contents of various compounds were determined.The results showed that the colloid contained a large amount of polysaccharide(64.92%)and protein(16.20%),and the content of small molecular compounds represented by PF(6.04%)was the lowest.2 Extraction,isolation and identification from GPThe proteins of SY Decoction,GC Decoction and SGD were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The results showed that there were two bands of alcohol precipitated protein in SGD,with molecular weights of 25-35 kDa and 63-75 kDa respectively,and the protein bands of 62-75 kDa were obvious.Both bands were derived from GC.And the GP was isolated and purified from GC by buffer cold soaking,alcohol precipitation,ion exchange chromatography and dialysis.The content of 63-75 kDa in GP was more than 85%.The isoelectric point(PI)of GP was determined by isoelectric focusing of full-column imaging capillary.The results showed that the PI of the main substances in GP were:5.52,5.57 and 5.62.The amino acid composition of GP were determined by amino acid automatic analyzer.The results showed that the contents of aspartic acid,serine and phenylalanine were relatively high The amino acid sequence of GP was obtained by high-resolution mass spectrometry and database.The protein is a lectin protein in GC.3 Construction and assembly mechanism of PF-GP self-assembled colloidal particlesTaking the binding amount of PF as the evaluation standard,five conditions of PF concentration,GP concentration,pH value,heating time and heating temperature were tested by single factor experiment.It was found that when PF and GP were mixed at a ratio of 1:10(pH=8)and heated at 100℃ for 40 min,the binding amount of PF(315.64 μg)was the largest,and the PF-GP self-assembly colloidal particles(NPs)was constructed.The physical and chemical properties of NPs were characterized by Dindal effect,laser particle size analyzer,scanning electron microscope(SEM)and TEM.It was found that PF had obvious Dindal phenomenon.The particle size of NPs was 148.23±0.0436 nm and the zeta potential was-45.85±9.1550 mV.SEM and TEM showed that the NPs were distributed in chains chain beads.The stability of NPs was evaluated.The results showed that the binding amount of PF,protein content and SDS-PAGE band of NPs did not change significantly after being placed at room temperature for 24 hours,stored at-80℃ for 15 days and repeatedly frozen and thawed at-20℃.The self-assembly mechanism of NPs was characterized by ultraviolet-visible absorption spectroscopy(UV),infrared spectroscopy(IR)and hydrogen nuclear magnetic resonance(1H-NMR).The UV results showed that after PF and GP formed self-assembled particles,the characteristic absorption peak at 210-260 nm of PF was disappeared or masked,which affected the UV absorption of benzene ring and carbonyl of PF.The IR results showed that after PF and GP formed self-assembled particles,the hydroxyl and carbonyl peaks of PF moved to the low-frequency direction,the peak shape of the aromatic hydrogen vibration peak on the benzene ring changed,and the skeleton vibration peak of benzene ring fluctuated or was covered up under the binding effect.The 1H-NMR results showeed that after PF and GP formed self-assembled particles,the hydrogen protons on the benzene ring of PF moved from high field to low field,the hydrogen protons at sugar sites and alicyclic sites also changed significantly,and new peaks appear in the low field region.The self-assembly mechanism of NPs was calculated and simulated by homology modeling,molecular docking and molecular dynamics.The molecular docking results showed that the hydrogen bond interaction(electrostatic interaction)and the hydrophobic interaction(van der Waals interaction)were the main binding forces between PF and GP.The molecular dynamics results showeed that the colloidal particles formed by PF and GP were relatively stable,and the binding site was more rigid.The binding energy of PF and GP was-16.88±0.95 kcal/mol.The binding energy decomposition showed that electrostatic energy and van der Waals energy were the main contribution energy,which was consistent with the conclusion of molecular docking.The residue analysis results showed that the GLY-19 residue with the strongest contribution can form two hydrogen bonds with PF.4 Safety and in vitro uptake of PF-GP self-assembled colloidal particlesThe safety of NPs was investigated through CCK-8 and hemolysis experiment.The CCK-8 results showed that NPs had no obvious toxicity to rat dorsal root ganglion cells(DRG).The hemolysis results showed that GP had partial hemolysis,while after formed colloidal particles with PF,there was no hemolysis.GP and NPs were labeled with fluorescein isothiocyanate(FITC),and the entry into A549 was observed by confocal laser scanning microscope.The results showed that both GP and NPs colloidal particles could enter cells,and the NPs entered cells more.ConclusionSGD is mainly composed of small molecular compounds,polysaccharides and proteins.PF is the most abundant in small molecular compounds.The protein with higher content in the decoction is mainly derived from GC.The GP isolated and purified is a lectin-like protein,which can form stable colloidal particles through self-assembly with PF.The particles have good biocompatibility and can enter cells,This suggests that GP is an effective carrier of PF,and explained the material basis of the compatibility between SY and GC. |
PDF Full Text Request