Studies On Phenolic Components And Biological Activities Of Mongolian Oak(Quercus Mongolica) Cups And Anticancer Mechanism Of Urolithin C

Mongolian oak(Quercus mongolica L.)is a kind of deciduous tree,which belongs to the genus Quercus in the Castanea family and are used for windbreak,fire-prevention and water conservation forests.The Dictionary of Medicinal Plant records that oak cup is astringent,warming and nontoxic with functions of astringency and hemostasis.However,oak cups,the abundant outer layer around the acorn,are discarded as waste,thus leading to huge resource waste and environment pollution.Therefore,it is great significance to study the active components and bioactive activities of oak cups as well as their potential values.Moreover,it has been reported that Mongolian oak is rich of ellagic acid(EA).EA with poor bioavailability in the gastrointestinal tract,could be extensively metabolized to urolithins,such as urolithins A,B,and C(Uros A,B,and C),in the colon.Therefore,it is essential to explore the real active compounds responsible for the health benefits after consuming the ellagintannins-containing foodstuffs.In the present study,the phenolic components and biological activities of oak cups,and the anticancer mechanism of Uro C were investigated.The main results were as follows:(1)In the present study,we optimized the best extract solvent and isolated the best crude extract with HPD-100 macroporous resin chromatography to obtain four fractions(fractions Ⅰ-Ⅳ,Frs Ⅰ-Ⅳ).Composition analysis through total phenols,flavonoids and tannins,condensed tannins,HPLC profiles and antioxidant assessment by DPPH,ABTS,reducing power,ORAC and CAA were performed to evaluate oak cups crude extracts and Frs Ⅰ-Ⅳ.In addition,the correlation between bioactive component and antioxidant activity were analyzed.It showed that 50%ethanol aqueous was the best extract solvent and HPD-100 macroporous resin could be developed as a simple and effective procedure for preparation of active components from oak cups.Among the optimal 50%ethanol crude extract(ECE)and its subsequent four fractions,Fr.Ⅱ possessed the largest elution rate and strongest antioxidant ability.The composition and antioxidant activities of Fr.Ⅱ and Fr.Ⅲ implied that composition of condensed tannins as well as its content might take dominant role in antioxidant ability of oak cups.HPLC exhibited that great enrichment of EA in Fr.Ⅲ and the compositions of phenolics were the main influencing factor to antioxidant ability.Moreover,the correlation analysis indicated that flavonoids were the strong antioxidant contributor in oak cups.(2)UPLC-QTOF-MS/MS exhibited that there were 24 phenolics in oak cups,and EA derivatives and kaempferol derivatives were the main phenolic components in oak cups.The acid hydrolysis experiment also exhibited that oak cups contained considerable EA existing mainly as ellagitannins(ETs)but not gallotannins.EA,ECE and Frs I-IV all showed strong inhibition on a-glucosidase and a-amylase,and exhibited much more drastic inhibitory activities against a-glucosidase than a-amylase.Furthermore,inhibition of ECE and EA on a-glucosidase were reversible and noncompetitive.Kaempferol derivatives showed stronger a-amylase inhibitory activity,while EA derivatives exhibited better inhibition against a-glucosidase.(3)Formation of advanced glycation end-products(AGEs),fructosamine adducts and a-dicarbonyl compounds were inhibited differently by EA,ECE and Frs I-IV,especially EA,Frs II and III.MTT assay exhibited that EA,ECE and Frs I-IV affected cell viability differently and Fr.IV might contain component with high anticancer activity.Thus,EA derivatives exhibited superior inhibition against glycation while kaempferol derivatives showed potent anti-cancer activity.(4)Anti-proliferative activities of EA,Uros A,B,and C in PC 12 cells,and the anticancer mechanism of Uro C were investigated.MTT assay showed that EA significantly promoted,while Uros inhibited the proliferation of PC12 cells at high concentrations,among which Uro C showed the strongest anti-proliferation.Uro C exerted inhibitory activity against cell proliferation through both apoptotic induction and S phase cell cycle arrest.In addition,Uro C treatment actively increased the lactate dehydrogenase release and malondialdehyde generation,stimulated reactive oxygen species formation and caused calcium dyshomeostasis,and imbalance in the Bcl-2/Bax ratio.Consequently,Uro C induced mitochondrial membrane depolarization,which triggered the caspase cascade,thereby shifting the balance in favor of apoptosis as evidenced by western blotting and real-time PCR.These observations demonstrated that Uro C might induce cell apoptosis through a mitochondria-mediated pathway.