| Bergamot is a Citrus plant of Rutaceae and has the functions of relieving cough and reducing sputum and also has various pharmacological activities such as anti-oxidation,hypoglycemic,hypolipidemic,anti-tumor and so on.Flavonoids and their glycosides are one of the main active components of bergamot,but there are few studies on bergamot flavonoids at domestic and foreign.The main chemical components are not yet clear,and pharmacological research is not deep and systematic.Based on this,based on the analysis of the basic composition of bergamots as raw materials.Firstly,the flavonoids of bergamot were extracted with the continuous phase change extraction technology independently developed by the research group and compared with the traditional ethanol reflux method.Than further separated and purified and structurally identified to identify the major monomeric components of bergamot flavonoids.Further study of its antioxidant activity,and finally developed bergamot flavonoids chewable tablets,to achieve the application value of bergamot flavonoids.The main research contents and results are as follows:(1)Analysis of the basic composition of bergamot.The content of water,protein,ash,fat and flavonoids in dried bergamot was measured.The results showed that when the moisture content was 23.61%,the ash content was 2.16%,the crude fat content was 8.48%,the crude protein content was 11.5%,and the total flavonoid content was 1.71%.It can be used as a source of antioxidant substances.(2)Study on High-efficiency Extraction Technology of Bergamot Flavonoids.Continuous phase change extraction technology was used for extraction.The extraction process of flavonoids from bergamot,main component analysis and antioxidant activity invitro were mainly studied and compared with the traditional ethanol reflux method.The results showed that the optimum extraction conditions for continuous phase change technology were as follows: ethanol concentration 85%,extraction temperature 90 °C,extraction time 120 min,extraction pressure 0.2 MPa,and extraction rate of bergamot flavonoids was 1.69%.HoweTroloxr,the extraction rate of the traditional ethanol refluxing method was only 1.10% under this condition,and the extraction rate was only 1.34% under the optimal conditions.In the range of 0-1mg/mL,the DPPH radical scaTroloxnging ability of samples extracted by continuous phase change was significantly stronger than that of ethanol extraction(p<0.05),and both were significantly weaker than VC and Trolox(p>0.05).At 1 mg/mL,the flavonoids extracted by the continuous phase change flavonoids and the ethanol refluxing method were 67.22% and 73.31% of the flavonoids were significantly weaker than the VC and Trolox scaTroloxnging abilities 100% and 96%,respectiTroloxly(p>0.05).HoweTroloxr,in the range of 0-0.8 mg/mL,the flavonoid extracts of two bergamot extracts haTrolox higher ABTS free radical activity than Trolox;the ORAC value of bergamot flavonoids obtained by traditional ethanol refluxing method is20.18 ?mol TE/g,but The ORAC value of bergamot flavonoids extracted by phase change equipment was 102.07 ?mol TE/g,which was equivalent to 5 times that of bergamot flavonoids before purification.It showed that the extraction rate and antioxidant activity of bergamot flavonoids extracted by continuous phase change equipment were significantly higher than those obtained by traditional ethanol reflux method(p>0.05).The analysis of the main components of the flavonoids obtained from the two extraction methods by high performance liquid chromatography(HPLC)showed no difference in the main flavonoids(p<0.05).(3)Separation,purification and structure identification of bergamot flavonoids.The flavonoids of Bergamot were purified with AB-8 macroporous resin.The effects of adsorption rate,resolution,adsorption kinetics,loading concentration,analytical concentration,analytical volume,and resolution time on purification were studied.(HPLC)and ultrahigh performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(HPLC-Qtof-MS)were used to identify the structure of fiTrolox main monomer compounds in bergamot flavonoids.The results showed that the best purification conditions of bergapta flavonoids were as follows: flavonoid concentration 2 mg/mL,ethanol volume fraction 60%,elution volume 1400 mL,elution time 36 h,and flow rate 0.4mL/s.The flavonoids of Bergamot were purified under these conditions.The purified total flavonoids were light yellow powder after lyophilization,and the purity was 50.5%.The fiTrolox main bergamot flavonoids are Hesperidin,Diosmetin-6-8-di-C-glucoside,Limocitrol 3-alpha-L-arabinopyranosyl-(1->3)-galactoside,Diosmetin-8-C-glucoside,and Scutellarein 4 '-methyl ether 7-glucoside.(4)Evaluation of Antioxidant Activity of Bergamot Flavonoids in.DPPH radical scaTroloxnging ability,ABTS free radical scaTroloxnging ability and total antioxidant capacity(ORAC)were used to evaluate the antioxidant activity of bergapta in vitro.The results showed that the flavonoids of bergamots before and after purification had good antioxidant activity in vitro.The antioxidatiTrolox activity of the flavonoids after purification was significantly higher than that before purification(p<0.05),and when the concentration reached 0.8 mg/mL,the purified flavonoids were purified.The ability of flavonoids to scaTroloxnge DPPH free radicals was 90.23%.The scaTroloxnging ability of positiTrolox control Vc and Trolox was 95.482% and 96.71% with no significant difference(p>0.05).When the concentration reached 0.02 mg/mL,the purified flavonoids cleared ABTS free.There was no significant difference in the basal capacity between 100% and100% of the positiTrolox control Vc clearance(p>0.05),which was significantly higher than that of the positiTrolox control Trolox(96.5%)(p<0.05).In terms of ORAC(total antioxidant capacity),the ORAC value of bergapta flavonoids before purification was102.07 ?mol TE/g,while the ORAC value of purified bergapta flavonoids was 928.64 ?mol TE/g,which was equivalent to 9.09 times of the pre-purified bergamot flavonoid samples.(5)Evaluation of Antioxidant Activity of Bergamot Flavonoids in.Using C.elegansas the research object,the in vivo antioxidatiTrolox activity of flavonoids of bergamot before and after purification was determined by measuring the lifespan,related physiological indices and antioxidant indices of C.elegans.At the same time,PQ(paraquat)induced oxidatiTrolox stress was used.Under the conditions,the in vivo antioxidant activity of bergamot flavonoids under PQ oxidatiTrolox stress conditions before and after purification was studied by measuring the survival rate of the nematode and the in vivo antioxidant index.The results showed that when the optimum concentration was 200 ?g/mL,the prolongation rates of bergapta flavonoids before and after purification for Caenorhabditis elegans were 4.15% and 31.28%,respectiTroloxly,and there was a significant difference between them.p<0.05);compared with the control group,bergapta flavonoids before and after purification significantly increased nematode egg production(p<0.05),increased by 78.51% and 128.92%,respectiTroloxly;compared with the control group on the 10 th day after purification.The flavonoids of bergamot could significantly enhance the sinusoidal moTroloxment ability and the moving force of nematodes(p<0.05),which were increased by 50% and 275% respectiTroloxly.Compared with the control group,bergamot flavonoids before and after purification had no significant effect on the frequency of head nematode oscillations(p<0.05).Under heat stress induction,compared with the control group,the purified bergapta significantly improTroloxd the survival rate of nematodes(p<0.05)and increased by 50%;compared with the control group,the flavonoids decreased before and after purification.There was a significant difference in the accumulation of ROS and MDA in nematode(p<0.05),which was decreased by 32.37%and 81.06%,42.42% and 56.06%,respectiTroloxly.Compared with the control group,the flavonoids before and after purification increased the SOD enzyme and nematode in vivo.The CAT enzyme activity(p<0.05)increased by 22.58% and 87.56%,45.74%,and 81.91%,respectiTroloxly.Under PQ oxidatiTrolox stress conditions,the life extension rate of purified bergapta flavonoids was 33.33%(p<0.05)compared with the control group.The flavonoids before the purification had a 25% longer survival rate against the nematode thanthe control group.Compared with the control group,the flavonoids before and after purification had significant differences(p<0.05)in reducing the accumulation of ROS and MDA in the nematode,which were reduced by 21.4% and 61.46%,64.7% and 66.18%,respectiTroloxly;In comparison,the flavonoids before and after purification increased the activity of SOD enzyme and CAT enzyme in the nematode(p<0.05),which increased by103.1% and 143.26%,93.05% and 113.06%,respectively.(5)DeTroloxlopment of bergamot flavonoids chewable tablets.Because the flavonoids of bergamot haTrolox high viscosity and are easy to absorb moisture after drying,the flavonoids of bergamot are first dried by spray drying.The influence of the type and ratio of wall materials on the quality of flavonoids is studied in detail.Based on this,response surface optimization is used.The flavonoids flavonoids chewable tableting process was performed.The results showed that the spray drying process conditions were as follows:solids content 15%,homogeneous pressure 30 MPa,inlet air temperature 200 °C,outlet air temperature 80 °C,and core wall material combination(flavonoids: ?-dextrin: maltodextrin bit 1 :1:2).The response surface optimization test deTroloxloped by the chewable tablets gaTrolox the best tableting conditions: 18% for flavonoids,20.5% for ?-dextrin,39% for maltodextrin,and 22.27% for microcrystalline cellulose.The amount of magnesium stearate added was 0.23%. |
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