| Deep eutectic solvent(DES), a eutectic mixture, is generally composed of hydrogen bond acceptor and hydrogen bond donor that are capable of associating with each other through hydrogen-bond interactions. They have merits such as low vapor pressure, nonflammability, simple preparation, no subsequent purification, and low price. DESs have been used in many fields, such as organic reactions, electrochemical, nanoparticles, biocatalysis and drugs. However, only a few studies have focused on the use of DESs for the extraction of bioactive compounds. The current research in the physicial and chemical properties of DESs is not systematical enough and their biocompatibility and biodegradability are rarely reported. This may limit their widespread applications. As a result, choline chloride and natural compounds are chosen as hydrogen bond acceptor and hydrogen bond donors respectively in this study to prepare various DESs. The physical and chemical properties were characterized and their biocompatibility and biodegradability were assessed systematically, and then they were applied to extract the bioactive flavonoids compound rutin. After separation and purification, the antioxidant activity of rutin were evaluated.Firstly, twenty kinds of eutectic solvents based on choline chloride eutectic were synthesized using amines, polyalcohols, organic acids, and sugars. The yield got 100% and the purity was more than 98%. The thermodynamic properties of these DESs were examined by differential scanning calorimetry instrument. They were clear, bright, uniform and stable liquid at room temperature and their melting point were all below 28℃. The water contents of most DESs were <3.8 wt%, except DESs whose hydrogen bond donor were hydrated acids or water-added DESs. The viscosities of prepared DESs at 30℃ were between 0.044 Pa ? s and 66.441 Pa ? s. Their conductivity ranged form 14.3 μS ? cm-1 and 9370 μS ? cm-1 at 30℃. The density lay between 1.0410 g ? cm-3 and 1.3313 g ? cm-3 at 30℃. The viscosity, conductivity and density of DESs were related to the degree of internal hydrogen bond network structure. In other words, the types and structures of various hydrogen bond donor were responsible for the physical and chemical properties of the solvents. As the temperature increased, the viscosity and density decreased while the conductivity increased.Secondly, the biocompatibility of the tested DESs was evaluated qualitatively and quantitatively by agar diffusion and dilution method and its biodegradability was assessed by Closed Bottle Test. The results are as follows. The amine-, alcohol- and sugar-based DESs did not inhibit bacterial growth, while the organic acid-based DESs had a significant inhibitory effect. Gram-negative bacteria were more sensitive than Gram-positive bacteria to acid-based DESs. In a word, the toxicity of DESs was related to the type and structure of hydrogen bond donor and the pH vaule of the solvents. The MIC and MBC values of Staphylococcus aureus ATCC 14128, Listeria monocytogenes ATCC 19115, Escherichia coli ATCC 8739 and Salmonella enteritidis ATCC 29213 were over 12 mmol/L and 16 mmol/L,especially the ChCl/p-toluenesulfonic acid, whose MIC and MBC values got 30 mmol/L and 50 mmol/L toward Listeria monocytogenes respectively. All DESs were environmentally friendly solvents with biodegradation >69.3%, classified as “readily biodegradable” after the 28 days of degradation biological experiments.In order to expand applications of deep eutectic solvents, this paper attempts to extract rutin form Sophora japonica using these solvents. The best solvent is ChCl/triethylene glycol in all prepared deep eutectic solvents. Single factor experiments showed that the optimum extraction conditions were: 270.3 mg rutin was extracted form 1.0 g S. japonica powder in 10 mL ChCl/triethylene glycol solvent with 20% water content and 1:4 component mole rate at 65℃ for 20 minutes. The extraction rate was 96%. After the optimization of extraction conditions with single factor and Box-Behnken Design, the optimum water content, extraction time, extraction temperature, the liquid-solid ratio and the molar ratios of solvent component were found to be 18%, 28 min, 70℃, 10 mL/1 g and 1:4. Under the above-mentioned conditions, the extracted amount of rutin reached 279.8 mg/g, close to the theoretical prediction value of 272.5 mg/g, illustrating that BBD experiment was a good fit model. Extracted sample solution was separated by macroporous adsorption resin and after purification and refinement the recovery of the extracted rutin attained 62.7%. The recovered product was definitely rutin by NMR spectrometer and achieved 95.5% purity. According to the antioxidant analysis of the obtained rutin, the IC50 of DPPH scavenging, O2- scavenging and ?OH scavenging were 5.68 μg/mL, 0.19 mg/m L and 0.28 mg/m L, respectively, indicating that rutin had excellent antioxidant activity and it was an admirable free radical scavenger.This work could provide some guidance to the rational design of new green solvents. It can not only promote the development of green extraction of natural bioactive substances, but also expand the applications of deep eutectic solvents. |
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