Separation And Purification Of Flavonoids From Citrus Fruits Of Different Varieties And Related Activities

China is one of the origins of citrus plants,with a long history of citrus cultivation and abundant resources.Citrus is popular among consumers because of its unique flavor and nutritional value.Because of its various bioactive and pharmacological functions,research on characteristic citrus nutrition and health has received increasing attention.Studies have shown that the bioactivity of citrus fruits is closely related to their rich bioactive substances,especially flavonoids.In this thesis,we conducted a study on the identification of citrus fruit flavonoids,the construction of separation and purification systems and the evaluation of chemical antioxidant ability and antitumor activity.The main results were as follows:1.A method was constructed for the enrichment of flavonoid segmental fractions of different citrus varieties based on solid-phase extraction technology.The representative varieties of pomelos,grapefruits,sweet oranges,bitter oranges,mandarins and tangerines were selected as raw materials,and the most suitable loading volume,elution concentration and elution volume of each citrus varieties were explored after ultrasonic-assisted extraction for segmental enrichment.After enrichment by solid-phase extraction,the segmented fractions of flavone-based fraction Ⅰ,flavanone-based fraction Ⅱ and polymethoxyflavone-based fraction Ⅲwere obtained respectively.The recoveries of fraction Ⅰ were all above 50.45%,fraction Ⅱ were all above 75%,and fraction Ⅲ were all above 46.55%.2.A high-purity purification method based on semi-prep HPLC for bioactive monomers of different citrus varieties was established.Based on the segmental enrichment by solid-phase extraction,the flavonoid extracts of different citrus varieties were further purified by semi-prep HPLC,and finally 2 flavones,8 flavanones,5 polymethoxyflavones and 3 coumarin monomers were obtained.The flavonoids from the pomelo variety ‘Yuhuan Wendan’ and purities were:vicenin-2(98.72%),rhoifolin(95.78%)and naringin(99.56%);the flavonoids from the grapefruit variety ‘Huyou’ and purities were: narirutin-4’-O-glucoside(98.42%),erocitrin(98.74%)and neoeriocitrin(99.17%);the flavonoid from the sweet orange variety ‘Lunwan Qicheng’ and purity was: narirutin(98.12%);the flavonoid from the tangerine variety ‘Ponkan’and the purity was: hesperidin(98.00%);the coumarins from the bitter orange variety ‘Daidai’and purities were: xanthotol(95.80%),meranzin(96.99%)and isomeranzin(97.24%);the flavonoids from the mandarin variety ‘Ougan’ and purities were: neohesperidin(98.56%),poncirin(98.03%),isosinensetin(98.76%),sinensetin(98.77%),nobiletin(99.15%),tangeretin(99.20%)and 5-demethylnobiletin(97.63%).3.The chemical antioxidant abilities of three segmented fractions extracted from 6 citrus varieties rich in flavones,flavanones and polymethoxyflavones and 18 purified monomers were evaluated and found to be highly variable.The flavone-based fraction and flavanone-based fraction showed strong antioxidant ability,while the polymethoxyflavone-based fraction showed poorer antioxidant ability.For the purified monomers from citrus,the antioxidant abilities of neoeriocitrin,eriocitrin,xanthotol,hesperidin and neohesperidin were stronger,and poorer for nobiletin,tangeretin and 5-demethylnobiletin.4.The antitumor activity of flavonoid segmented fractions and purified monomers from six citrus varieties was evaluated and found that different fractions and monomers have inhibitory effects,but with different inhibition rates.The polymethoxyflavone-based fraction inhibited the proliferation of human cervical cancer cells(Hela)and human breast cancer cells(MDA-MB-231)more,while the other fractions had weaker inhibitory effects.Most of the purified monomers showed inhibitory effects on tumor cell proliferation with a concentration-dependent trend,however Hela cells were more sensitive than MDA-MB-231 cells.Among the 18 monomers,xanthotol,meranzin,isomeranzin,nobiletin,tangeretin and5-demethylnobiletin showed stronger inhibitory effects on Hela and MDA-MB-231 cells.In this study,the flavonoid separation and purification system based on different varieties of citrus was first established,and a total of eighteen monomers were purified from six citrus varieties.For the first time,vicenin-2 was purified from pomelo varieties,narirutin-4’-O-glucoside and eriocitrin were purified from grapefruit varieties,xanthotol was purified from bitter orange,and isomeranzin was purified from mandarin varieties,which can be used to study the antioxidant and antitumor activities and mechanisms of citrus flavonoids,and provide the preliminary technology reserve for the development and utilization of these bioactive substances.