| Deep eutectic solvent is a kind of homogeneous stable system formed by hydrogen bond donor and hydrogen bond acceptor by a certain stoichiometric ratio.As a new type of ionic liquid,choline-based eutectic solvent has more prominent advantages,such as low toxicity,100% atom utilization,biodegradable and low price etc.Therefore,it is widely used in the fields of synthesis,catalysis,electrochemistry,separation and extraction of traditional Chinese medicine with its unique physical and chemical properties.In this paper,salvianolic acid B,one of the main water-soluble active components of Radix Salviae miltiorrhizae,were selected as the experimental object,the stability,extraction and pharmacokinetics of salvianolic acid B dissolved in deep eutectic solvent were studied.The main work of this paper is as follows:(1)The stability of salvianolic acid B was investigated in four kinds of benign choline-based deep eutectic solvent modified with different hydrogen-bond donors(ethylene glycol,1,2-propanediol,glycerol and 1,4-butanediol),and the effect of water content in deep eutectic solvent,temperature,heating time for the stability of salvianolic acid B was also studied.The degradation products of salvianolic acid B were analyzed by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry(LC-MS/MS).Moreover,the mechanism of improving stability of salvianolic acid B in deep eutectic solvent was also discussed by analyzing the content variation trends of degradation products.Obviously,the stability test demonstrated that salvianolic acid B was more stable in deep eutectic solvent than in water or ethanol solution at room temperature or high temperatures.And choline chloride-glycerol(molar ratio 1: 2)was optimized to offer a satisfactory enhancement effect for the stability of salvianolic acid B.The optimum experiment proved that the stabilizing capacity of deep eutectic solvent suffered major influence from the water content in deep eutectic solvent solution,minor influence from the structure of hydrogen-bond donors and minimal influence from the molar ratio of quaternary ammonium salts to hydrogen-bond donors.(2)Twenty-five kinds of benign choline chloride-based deep eutectic solvent with microwave-assisted methods were applied to quickly extract active components(rosmarinic acid,lithospermic acid,salvionalic acid B,salvionalic acid A and tanshinone IIA)from Radix Salviae miltiorrhizae.The extraction factors,including the composition,ratio of HBA/HBD and water content of deep eutectic solvent,temperature,time,power of microwave,and solid/liquid ratio,were investigated systematically by single factor and response surface methodology.The hydrophilic and hydrophobic ingredients were extracted simultaneously under the optimized conditions: 20 vol% of water in choline chloride/1,2-propanediol(1: 1,molar ratio)as solvent,microwave power of 800 W,temperature at 70?C,time at 11.11 min,and solid/liquid ratio of 7 mg/m L.The extraction yield was comparable to,or even better than,conventional methods with organic solvents.The microstructure alteration of samples before and after extraction was also investigated,and plant cells were thoroughly ruptured when extracted by deep eutectic solvent with microwave-assisted methods,this might be one of the important factors of the high extraction efficiency.(3)The toxicity of deep eutectic solvent was analyzed by acute toxicity test using ChCl-GL(1: 2)as the test solution,and ChCl-GL(1: 2)was administered orally to mice.And the median lethal dose(LD50)was estimated though the Karber method.According to the Karber method,the estimated LD50 was 7733 mg/kg with a 95% confidence interval of 7130-8387 mg/kg for oral administration.This indicates that ChCl-GL(1: 2)is non-toxic,and is relatively safe on clinical application for oral dosage forms.(4)With salvianolic acid B as a model drug,the feasibility of deep eutectic solvent as a drug carrier for oral preparation was investigated by evaluating the influence of deep eutectic solvent on the pharmacokinetics of salvianolic acid B.To comparison the difference of pharmacokinetics between salvianolic acid B dissolved in ChCl-GL(1: 2)and in water,a rapid and sensitive ultra-performance liquid chromatography coupled with mass spectrum was established to determine salvianolic acid B and its metabolites in rat plasma.The drug-time curve of salvianolic acid B dissolved in different solvents had the similar change tendency basically.Compared to water,the pharmacokinetic parameters clarified that ChCl-GL(1: 2)could promote the absorption of salvianolic acid B,the peak concentration(Cmax)of 0.308 ± 0.020 mg/L was slightly higher than 0.277 ± 0.024 mg/L(salvianolic acid B dissolved in water),and the peak time(Tmax)was significantly decreased from 30 min(salvianolic acid B dissolved in water)to 20 min.There was no significant difference on the metabolites between salvianolic acid B dissolved in ChCl-GL(1: 2)and in water.(5)To investigate the effect of deep eutectic solvents on the tissue distribution of salvianolic acid B in mice.The fasted mice were randomly divided into control group and test group,which were oral administration of salvianolic acid B dissolved in water and salvianolic acid B dissolved in ChCl-GL(1: 2),respectively.After intragastric administration of salvianolic acid B,the main tissues(heart,spleen,lung,kidney,brain and liver)were collected at different time points.The content of salvianolic acid B was determined by UPLC-MS.The results shown that compared with the control group,deep eutectic solvent had certain effects on tissue distribution of salvianolic acid B in mice,including: the peak times of of salvianolic acid B in liver in test group and lung were both 20 min,which were 10 min shorter than the control group.ChCl-GL(1: 2)was of great important for promoting the distribution of salvianolic acid B in liver,and the peak concentration was 1.94 ± 0.23 ?g/g,which was significantly higher than the control group(1.02 ± 0.05 ?g/g).However,ChCl-GL(1: 2)had no effect on the distribution of salvianolic acid B in kidney.In conclusion,deep eutectic solvent could promote the membrane permeation of salvianolic acid B,accelerate the absorption and elimination,but shown less advantage of the absorption of salvianolic acid B in lung and kidney.In addition,the content of the two groups of salvianolic acid B in the brain was also lower than limit of detection,which only be obtained by spectroscopy and mass spectrometry;and the two groups were not detected salvianolic acid B in the heart and spleen.The above results may be able to provide some reference for the subsequent experimental study and clinical application of deep eutectic solvent. |
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