| Limonin,one of the most important active compounds in citrus fruits,is found primarily in citrus seeds and is widely used in the pharmaceutical,food,and agricultural domains owing to its anti-tumor,anti-inflammatory,insecticidal,and other activities.The extraction and isolation of limonin from citrus seeds is an important part of the development and utilization of citrus resources,whereas one of the key issues that must be addressed in pre-industrial research is the search for a green,environmentally friendly,efficient,and reasonably priced extraction method.Limonin’s poor water solubility limits its use in food and pharmaceuticals,and to use of nanoencapsulation to delay the release of limonin could be a viable solution to the issue its of utilization.Additionally,there is a lack of traceable limonin purity certified reference material and matrix reference materials in related fields.It is difficult to distinguish between the accuracy and reliability of the results because the same measures cannot be compared across various nations,laboratories,and personnel.As a result,certified limonin reference material with good metrological properties is crucial for related analysis.Based on the foregoing,this study optimized the extraction process of limonins from citrus seeds using natural deep eutectic solvents（NADESs）.The high-purity limonin monomer was purified by adsorption on macroporous resin combined with antisolvent crystallization,laying the foundation for further researches.The purity reference material of limonin was prepared and their homogeneity and stability were assessed following pertinent technical specifications of national reference materials.Finally,the purified limonin was microencapsulated and its release characteristics and antioxidant activities were investigated in vitro during simulated gastrointestinal digestion.The aforementioned research offers fresh approaches and ideas for citrus limonin extraction,purification,and microencapsulation.It also provides data support and a scientifically valid foundation for the development and utilization of citrus resources.The main contents are as follows:（1）Optimization of extraction process of citrus limonins based on natural low eutectic solventUsing choline chloride as the hydrogen bond acceptor and four different types of hydrogen bond donors（organic acids,polyols,sugars,and amines）in a certain molar ratio,13 NADESs were synthesized.The total yield of three limonins—limonin,nomilin,and obacunone was used as the evaluation index,and ultrasound-assisted NADESs were used to extract limonins from citrus seeds.The results showed that the best extraction was accomplished when choline formic acid chloride（1:4,n/n）was used as the solvent.On this foundation,the response surface optimization method was used to determine the optimal extraction conditions for the extraction of limonins from citrus seeds,with water content,liquid-solid ratio,extraction time,and extraction temperature as the primary influencing factors:12%（v/v）,liquid-solid ratio,10m L/g,extraction time,34 min,and extraction temperature,60°C.Moreover,limonin monomer with a purity of 98.35%was obtained after the separation and purification of limonin utilizing adsorption on the macroporous resin in conjunction with the antisolvent crystallization technique.（2）Development of limonin purity certified reference materialStructure validation of limonin samples by nuclear magnetic resonance spectroscopy（1HNMR）,Fourier transform infrared spectroscopy（FTIR）,and mass spectrometry（MS）,and then purified by recrystallization method to prepare limonin purity reference material.The parameters of the high performance liquid chromatography（HPLC）column,mobile phase,sample concentration,and detection wavelength were investigated based on the response of limonin and its impurities.The optimal parameters were as follows:column,SB-C18（250 mm4.6 mm,5.0 um）;mobile phase,acetonitrile:water=38:62;flow rate,1 m L/min;sample concentration,500 mg/L;column temperature,30°C;injection volume,10 L;detection wavelength,210 nm.The purity of limonin improved from 98.01%to 99.48%after recrystallization.The mass balance method（MB）and the quantitative nuclear magnetic resonance（q NMR）,were used to determine samples of limonin reference material.The HPLC method measured 99.48%of the main components,0.3082%of moisture,0.0136%of residual solvent,and 0.0511%of inorganic impurities.The final determination of the limonin purity reference material was 99.12%,with the MB obtaining a value of 99.11%and the q NMR obtaining a value of 99.13%.Using HPLC,the homogeneity and stability of the reference material were investigated,and the development process level of uncertainty was assessed.The findings demonstrated the good homogeneity and stability of the limonin purity reference material,which had a purity determination of 99.12%,an extended uncertainty of U=0.43%,and a validity of 6 months.（3）Microencapsulation of limoninLimonin nano-microcapsules were prepared using chitosan（CS）as the carrier and sodium tripolyphosphate（TPP）as the cross-linking agent,with the focus on the effects of chitosan concentration,p H value of chitosan solution,and mass ratio of chitosan to sodium tripolyphosphate on microcapsule encapsulation rate.By conducting orthogonal experiments,it was possible to determine the optimum conditions for the production of limonin nano-microcapsules:CS concentration,1.5 mg/m L;CS solution,p H 4.5;CS to TPP mass ratio,5:1.The FTIR characterization showed that limonin was present in the limonin nano-microcapsules,indicating successful embedding of nano-microcapsules.The average particle size of nano-microcapsules of 293.75 nm,with an embedding rate of 48.97%,and a relatively uniform size distribution.The release of limonin nano-microcapsules was 9.59%lower than that of limonin at 5 h,and moreover,they can be released continuously,according to an in vitro digestion simulation.And its ability to scavenge DPPH free radicals was 1.14～1.38 times more than that of limonin itself. |
