| Caffeine is a purine alkaloid,which has the functions of relieving fatigue,exciting the nerve center,promoting blood circulation and improving mood.It is widely used in food and medicine and other fields.It naturally exists in the leaves and seeds of many plants,with the highest content in tea.At present,water phase Ⅰ,ethyl acetate phase Ⅱ and water phase Ⅱ(the raffinate)are obtainedin the industrial production of tea polyphenols through the ethyl acetate extraction-water extraction process.The raffinate contains a large amount of caffeine is often directly discharged as wastewater.In order to effectively and rationally use resources,reduce environmental pollution,and improve the efficiency of tea deep processing enterprises,it is necessary to establish an efficient and economical method to separate and purify caffeine in the raffinate.Therefore,the extraction process was analyzed and the purification process of caffeine from the raffinate by sublimation method was optimized.In order to provide a theoretical basis for the use of tea caffeine,the preventive effect of the purified tea caffeine on non-alcoholic fatty liver disease(NAFLD)was explored through animal experiments in vivo,the main research content and results are as follows:1.The analysis of main components in the raffinate,water phase Ⅰ and ethyl acetate phase Ⅱ and the distribution of the main chemical components of tea in the three components.The main chemical components in the raffinate were tea polyphenols,caffeine and soluble sugars,which account for 51.46%,22.85% and 10.43%,respectively.The ethyl acetate phase Ⅱ was mainly composed of tea polyphenols and other chemical components was extremely low,and the water phase Ⅰ was mainly composed of soluble sugars and amino acids.After the extraction process,the distribution proportions of tea polyphenols in the raffinate,water phase Ⅰ and ethyl acetate phase Ⅱ were 32.45%,23.75% and 43.81%,respectively.The distribution proportions of caffeine in the three components were respectively 63.33%,33.89% and 2.78%.More than 90% of the soluble sugars and amino acids were distributed in water phase Ⅰ.The more active ester catechins are mainly distributed in the ethyl acetate phase Ⅱ,while the non-ester catechins are mainly distributed in the raffinate.The results showed that the ethyl acetate extraction-water extraction process could realize the separation of the main active substances in tea and was suitable for the industrial production of tea polyphenols.The raffinate had complex chemical components and high caffeine content,so sublimation method was more suitable for the purification of caffeine.2.Screening of pretreatment agent for raffinate and optimization of process for purifying tea caffeine by sublimation method.The caffeine in the raffinate was separated and purified by sublimation method,and sodium carbonate was screened out as the pretreatment agent with higher recovery rate and purity of caffeine.The effects of p H,sublimation temperature,sublimation time and addition of sodium carbonate on the recovery rate of caffeine were explored,and the optimal sublimation process conditions were determined through response surface optimization experiments: the sublimation temperature was 214℃,the sublimation time was 28 min,and the mass ratio of sodium carbonate addition to solidified raffinate was 2.3:1(w/w),the recovery rate of caffeine under this condition can reach 76.21%,and the purity of caffeine is higher than 98.5%.The obtained sublimation process was used to separate and purify the caffeine from four different raw materials and the traditional sublimation method was used as a comparison,the results showed that the improved sublimation process was suitable for the separation and purification of caffeine in different raw materials,and the recovery rate was about 29% higher than that of the traditional sublimation method.The purified substance was confirmed to be caffeine by ultraviolet spectroscopy,mass spectrometry,and infrared spectroscopy,and the scanning electron microscope showed that the crystals of caffeine are needle-like and rod-like,with dense accumulation on the surface and high quality.3.Study on the effect of tea caffeine in preventing NAFLD induced by high-fat diet.A mouse NAFLD model was constructed by feeding mice with a high-fat diet,and the effects of different doses of tea caffeine under a high-fat diet on obesity,liver lipid accumulation,insulin resistance,liver oxidative stress,liver inflammation were compared and observed.Compared with the high-fat diet group,the tea caffeine prevention group can reduce the weight and white fat coefficient of mice,increase the brown fat coefficient;reduce the content of TG,TC and LDL-C in the serum of mice,and increase the content of HDL-C.Reduce the liver mass index and the content of TG and TC in the liver;improve insulin resistance and glucose tolerance;reduce the content of MDA in the liver of mice,and increase the content of SOD,CAT and GSH in the liver of mice;down-regulate the level of TNF-α and IL-6 in mouse serum,and up-regulate the level of IL-10;HE staining of liver slices showed that the tea caffeine reduced liver steatosis and the production of fatty vacuoles,thus has the effect of preventing NAFLD.4.The effect of tea caffeine on the expression of inflammation and fat metabolism-related genes in the liver of NAFLD mice induced by high-fat diet.The expression of related genes in the liver of mice was detected by q RT-PCR,and the results showed that tea caffeine can down-regulate the expression of pro-inflammatory factor TNF-α and up-regulate the expression of anti-inflammatory factor IL-10 in the liver of high-fat diet mice to prevent liver Inflammation,down-regulates the expression of fat synthesis-related genes SREBP-1c,FAS,ACC-1 and SCD1,and up-regulates the expression of lipolysis-related genes PPAR-α and CPT-1 to inhibit the accumulation of fat in the liver,So as to prevent NAFLD. |
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