Study On The Application Of Natural Deep Eutectic Solvents In The Extraction Of Saponins From Panax Notoginseng And The Bioactivities Of Their Solutions

Panax notoginseng（PN）（Burk.）F.H.Chen,a precious Chinese herbal medicine which has been used for more than 600 years.PN has the effects of removing blood stasis,stopping bleeding,diminishing swelling,easing pain,invigorating qi,promoting blood circulation,etc.and has been intensively applied to the prevention and treatment of cardiovascular and cerebrovascular diseases in China.Saponins are the main active components of PN,and also the main components of related preparations of PN used in the clinic.Modern pharmacological research has shown that saponins from PN have pharmacological activities such as anti-thrombosis,dilating blood vessels,anti-inflammation,anti-aging,anti-tumour and antioxidant activities.It is a hot topic in this research area over the last decades on how to extract them efficiently and safely.The existing extraction methods of saponins from PN typically are the traditional extraction methods such as heat reflux extraction,water extraction and alcohol precipitation,percolation extraction,etc.,and ethanol and water are mainly used as extraction solvents.However,these solvents have some defects,such as ethanol is flammable,low concentration of alcohol can easily cause gelatinization during extraction,and the extraction efficiency of water is relatively low.Therefore,the search for a green,natural,and effective solvent for the extraction of saponins from PN would not only have important value in the extraction process of PN,but also provide important references and ideas for the future development and application of PN and other Chinese herbal medicines containing saponin components.Nature Deep Eutectic Solvents（NADES）are binary or ternary eutectic mixtures formed by the combination of hydrogen bond acceptors（such as quaternary ammonium salts and zwitterions）and hydrogen bond donors（such as carboxylic acids,polyols,and sugars）at a certain stoichiometry,which are homogeneous mixtures in liquid state at room temperature.NADES is a new type of environmentally friendly solvent because their raw materials are all natural products,with low price and recyclability,which accords with the concept of green chemistry.In addition,NADES have got some advanced features,including excellent biocompatibility,better biodegradability,acceptable biotoxicity and sustainability.In recent years,NADES have been of interest in the field of extracting,separating and purifying active components from medicinal plants.Some NADES can form a large number of hydrogen bonds with target components,so NADES often show better extraction capability than traditional solvents.In this study,NADES was applied to optimize the extraction process of saponins from PN,and the cytotoxicity of NADES and the related bioactivities of saponins NADES extract were investigated.And the main results of research were as follows:1.Preliminary sreening of NADES:The literature survey firstly performed for this study,24 kinds of NADES suitable for extracting saponins were screened out.Then,14 NADES which were homogeneous and stable liquids at room temperature were obtained by preparation.2.Determination of NADES species,components molar ratio and water content:By determining of the solubility of saponins in above NADES,and investigating of the effects of the components molar ratio and water content of NADES on the extraction yield of saponins,using the solubility and extraction yield of saponins from PN were used as the evaluation index of the process.The results confirmed that the optimum NADES composed of 1:2（mol/mol）of choline chloride/urea with water content of 45%（v/v）for extraction saponins from PN.3.Extraction of saponins from PN by ultrasonic-assisted NADES and optimization of extraction parameters:Taking the extraction yield of saponins as the index.Ultrasound-assisted NADES has been used to extract saponins from PN,and the optimal extraction conditions were optimized by combining single factor and response surface experimental design.The optimal extraction conditions were as follows:the extraction temperature was 47℃,the extraction time was 36 min,and the liquid-solid ratio was 40 m L/g.Under these conditions,the extraction yield of saponins（71.97 mg/g）are at the same level as 70%（v/v）methanol（70.89 mg/g）and much better than that of water（65.60 mg/g）.4.Preliminary investigation on the recoverability of NADES:The saponins were initially recovered from NADES by HPD101 macroporous adsorption resin with a satisfactory yield of 84.57%,and the volum recovery yield of pure NADES was 75.76%.Subsequently,according to optimized extraction method,the extraction yield of saponins was 70.22 mg/g by using the recovered NADES for the second extraction,which was similar to that of the first extraction（71.97 mg/m L）.5.Study on cytotoxicity of NADES:Cytotoxicity of NADES on RAW264.7,HEK293 and A549 cells was investigated using the methyl thiazolyl tetrazolium assay and hematoxylin-eosin staining.Compared with the published studies on the cytotoxicity of ILs,NADES had a relatively high IC₅₀value,that was,it showed a relatively small cytotoxicity.6.Study on bioactivities of saponins NADES extract:（1）The anti-inflammatory activity results of saponins NADES extract indicated that saponins NADES extract had a greater inhibitory effect on the m RNA expression of inflammatory cytokines tumor necrosis factor-αand interleukin-1βthan water extract,and it showed a dose-dependent relationship.（2）The antioxidant activity results of saponins NADES extract indicated that the saponins NADES extract had a stronger ability to clear OH and ABTS free radicals than water extract.（3）The anticoagulant activity results of saponins NADES extract showed that only the concentration of 25 mg/m L saponins NADES extract significantly prolonged the activated partial thromboplastin time compared to the blank group（physiological saline）（P<0.05）,and the concentrations of 12.5 and 25 mg/m L saponins NADES extract more significantly prolonged the prothrombin time（P<0.01）,while other dosage groups were not statistically significant,which indicated that a certain concentration of saponins NADES extract had strong anticoagulant.In summary,the optimization of process conditions for ultrasound-assisted extraction of saponins from PN using NADES indicates that NADES can be an effective,green,and sustainable alternative to traditional organic solvents for extracting active saponin components from PN.In addition,NADES had significantly lower cytotoxicity compared to ILs,and the NADES extract containing saponins exhibited strong anti-inflammatory,antioxidant,and anticoagulant activities overall.These findings provide ideas and valuable references for the future application and innovative development of NADES in the field of traditional Chinese medicine pharmaceuticals.