Study On Extraction,Isolation,Purification And In Vitro Digestion Simulation Of Anthocyanins From Perilla Leaves

Perilla is an annual herb of Labiatae.It is the first batch of medicinal and edible homologous plants included by the Ministry of Health of China.Perilla leaves are rich in anthocyanins,which have antioxidant,antibacterial and hypoglycemic effects.They have good application prospects in food and drug raw materials,cosmetics raw materials and biological pesticides.In this thesis,perilla leaf was used as raw material,ultrasonic assisted acidification ethanol extraction of anthocyanins from Perilla leaves,the purification process of anthocyanins from perilla leaves by macroporous resin and the technology of separating anthocyanins from Perilla leaves by Sephadex LH-20 dextran gel were optimized.The anthocyanin components of perilla leaves were analyzed,and the changes of total phenols,antioxidant and hypoglycemic activities before and after simulated gastrointestinal digestion were studied.The following results were obtained:(1)Using acidified ethanol as extraction solvent,perilla leaf anthocyanins were extracted by ultrasonic assisted extraction method.Taking the extraction amount of perilla leaf anthocyanins as evaluation index,four single factor experiments(extraction temperature,ultrasonic time,ethanol volume fraction and liquid-material ratio)were carried out.On the basis of single factor experiment,the extraction process parameters were optimized by Box-Behnken response surface method.The results showed that the optimum extraction conditions were as follows:extraction temperature 50?,ultrasonic time 37 min,ethanol volume fraction 40%and liquid-solid ratio 16:1(mL/g).Under this condition,the extraction amount of anthocyanin from perilla leaves was 715.56 mg/100 g.(2)The purification effects of five macroporous resins(AB-8,DM301,D101,X-5 and NKA-II)on anthocyanins from perilla leaves were studied.Based on the comprehensive analysis of the static experimental results,AB-8 resin(adsorption rate 85.62%,desorption rate 80.44%)was determined to be the best resin for the purification of anthocyanins from perilla leaves.The dynamic adsorption-desorption experiment of extracted perilla leaf anthocyanins in AB-8 macroporous resin column was carried out.Through single factor experiment and response surface optimization design experiment,it was determined that the optimal adsorption conditions were loading concentration 4.0 mg/mL,loading solution pH2.0 and loading flow rate 2.0 mL/min.Under these conditions,the anthocyanin adsorption rate was 95.42%.The optimum desorption conditions were elution ethanol concentration84%,elution flow rate 1.5 mL/min and eluent pH2.5.Under these conditions,the desorption rate of anthocyanins was 96.42%.The anthocyanin crude extract from perilla leaves was purified by AB-8 resin under the best conditions.The anthocyanin color value was increased from 12.15 to 30.77.(3)Sephadex LH-20 dextran gel was used to separate and purify the anthocyanins from Perilla leaves after purification with AB-8 macroporous resin.The effects of loading concentration,eluent(methanol)concentration and elution flow rate on the separation and purification of perilla leaf anthocyanins were investigated.The optimum conditions for separation and purification were determined as follows:loading concentration of 20 mg/mL,eluent concentration of 50%methanol(pH=2.0),elution flow rate of 1.3 mL/min.Under these conditions,the separation effect of anthocyanins from perilla leaves was the best,and three component peaks were collected.The anthocyanin components of perilla leaves were preliminarily identified by HPLC-MS.Two anthocyanin monomers were determined:Cyanidin 3-O-caffeoylglucoside-5-O-glucoside(C₃₆H₃₇O₁₉)and Cyanidin 3-O-caffeoylglucoside-5-O-malonylglucoside(C₃₉H₃₈O₂₂).(4)The perilla leaves powder,perilla leaf anthocyanins extraction powder,first purified powder(macroporous resin)and second purified powder(dextran gel chromatography)were mixed with rice starch at 5%,10%and 15%respectively.The gelatinization indexes of different samples were determined by RVA-Tec Master.It was shown that the gelatinization properties of the samples are closely related to the addition of active substances.With the addition of active substances increasing from 5%to 15%,the peak viscosity,trough viscosity,final viscosity and setback value of RVA samples decreased significantly,and the gelatinization temperature increased.The RVA sample with 15%first purified anthocyanin powder had the lowest final viscosity of 1562.5 c P,the lowest setback value of 482.0 c P and the maximum breakdown value of 1722.5 c P.(5)The gelatinized samples were subjected to gastrointestinal simulated digestion experiment.The glucose equivalent of the samples during digestion was measured to analyze the inhibitory effect of perilla leaf anthocyanin on the sugar raising reaction of starch in vitro.The results showed that with the increase of the amount of active substances from 5%to 15%,the release of reducing sugar decreased significantly after digestion,and the area under the curve(AUC value)between glucose release and time showed a downward trend during digestion,which inhibited the sugar raising reaction in vitro.The content of polyphenols before and after digestion was determined by Folin Phenol method.The results showed that the content of total phenols increased significantly after gastrointestinal simulation digestion.The highest content of polyphenols after digestion was the sample added with 15%first purified anthocyanin powder,up to 81.04 mg GAE/100 g DW,which was 2.19 times higher than that before digestion.The results of DPPH and ABTS antioxidant experiments showed that the antioxidant capacity was significantly improved after gastrointestinal simulated digestion in vitro.The ability of scavenging DPPH free radicals after digestion was about 1.16?1.93 times that before digestion,and the ability of scavenging ABTS free radicals after digestion was about1.15?1.53 times that before digestion.