Efficient Extraction,Antioxidant And Antitumor Activities Evaluation In Vitro Of Polyphenols From The Leaves Of Quercus Dentata Thunb

As a kind of medicinal and edible plant resource,the leaves of Quercus dentata Thunb are mainly produced in north China,especially in the mountainous areas of Henan,Hebei,Shanxi,and Shandong provinces.Studies have shown that the leaves of Quercus dentata Thunb contain rich bioactive substances such as flavonoids and other polyphenols.However,there are no relevant studies on the extraction process,composition analysis,antioxidant and antitumor activities in vitro of its polyphenols.In this paper,the extraction process of polyphenol compounds was optimized by response surface design,the chemical composition of the extract was preliminically explored,and the antioxidant and antitumor activities in vitro were comprehensively evaluated,providing certain theoretical basis of the leaves of Quercus dentata Thunb for the systematic study.The main research contents are as follows:1.Study on the extraction technology of phenolic compounds from leaves of Quercus dentata ThunbThe ultrasonic-coupled deep eutectic solvents method was used for the first time to extract phenolic compounds efficiently from the leaves of Quercus dentata Thunb.With the total polyphenol content as the index,five factors including the type of deep eutectic solvent,water content,solid-liquid ratio,extraction time and extraction temperature were investigated.Combined with the response surface analysis test,the optimal extraction parameters were obtained,that is,the deep eutectic solvent composed of choline chloride and 1,4-butanediol（molar ratio 1:2）with water content of 38%was used as the extraction medium,and the solid-liquid ratio was 1:30g·m L^-1,the extraction time was 31 min,the extraction temperature was 55℃,and the total polyphenol content was 78.56±2.44 mg GAE·g^-1.In addition,scanning electron microscopy analysis showed that the cell structure of the extraction powder obtained by ultrasonic-coupled deep eutectic solvent was significantly damaged,resulting in more components entering the solvent,which may be one of the reasons for the high extraction efficiency of ultrasonic-coupled deep eutectic solvent.2.Preliminary qualitative and quantitative analysis of chemical constituents of polyphenol extract from leaves of Quercus dentata ThunbEleven phenolic compounds were identified by high performance liquid chromatography quadrupole/time-of flight mass spectrometry,mainly flavonol glycosides with quercetin,kaempferol,and isorhamnetin as aglycones.Among them,The first nine kinds were quercetin-3-O-galactoside,isoquercitrin,guaijaverin,astragalin,isorhamnosin-3-O-glucoside,kaempferol 3-O-[6"-O-（trans-p-coumaroyl）]-beta-D-glucopyranoside,kaempferol 3-O-[2"-O-（trans-p-coumaroyl）-6"O-acetyl]-beta-D-glucopyranoside,kaempferol 3-O-[2",6"-di-O-（trans-p-coumaroyl）]-beta-D-glucopyranoside,kaempferol3-O-（2’’-cis-p-coumaroyl-6’’-trans-p-coumaroyl）-beta-D-glucopyranoside.Compound 10 was kaemferol 3-O-[2",6"-di-O-（trans-p-coumaroyl）-3",4"-di-O-acetyl]-beta-D-glucopyranoside or kaempferol 3-O-（2’’,4’’-diacetyl-3’’-cis-p-coumaroyl-6’’-trans-p-coumaroyl）-beta-D-glucopyranoside,compound 11 was kaempferol3-O-（2’’,4’’-diacetyl-3’’-cis-p-coumaroyl-6’’-trans-p-coumaroyl）-beta-D-glucopyranoside or kaempferol 3-O-（2’’-trans-p-coumaroyl-3’’,4’’-diacetyl-6’’-cis-p-coumaroyl）-β-D-glucopyranoside,and their composition need further confirmation.At the same time,a HPLC method for simultaneous determination of five flavonoid glycosides in leaves of Quercus dentata Thunb was established.The components were ranked by the content as follows:guaijaverin<isorhamnosin-3-O-glucoside<isoquercitrin<quercetin-3-O-galactoside<astragalin.The corresponding contents were 0.29 mg·g^-1,0.34 mg·g^-1,0.91 mg·g^-1,1.12 mg·g^-1 and 1.31 mg·g^-1,respectively,which provided reference for further research on pharmacodynamic substance basis of leaves of Quercus dentata Thunb.3.Antioxidant and antitumor activities of polyphenol extract from the leaves of Quercus dentata Thunb in vitroThe antioxidant activity of the extraction was evaluated by 7 chemical methods in vitro.The extraction showed good antioxidant activity in reducing transition metals(Fe³⁺,Cu²⁺,Mo⁶⁺)and scavenging free radicals（DPPH·,ABTS⁺·,O₂^-·and·OH）.With the increase of the concentration of the extraction,the reduction ability of transition metal ions and free radical scavenging ability were also increased.Among them,the IC₅₀ values of scavenging DPPH·and ABTS⁺·free radicals were 10.87±0.25μg·m L^-1 and 8.04±0.24μg·m L^-1,whose activities were similar to that of Vc(IC₅₀=9.20±0.23μg·m L^-1 and 6.38±0.25μg·m L^-1,p>0.05).The IC₅₀ values of scavenging O₂^-·and·OH free radicals were 149.47±5.27μg·m L^-1 and 131.73±5.31μg·m L^-1,which were higher than Vc(IC₅₀=86.60±2.05μg·m L^-1 and 92.69±2.81μg·m L^-1,p<0.05).On the other hand,antioxidant activity of the extraction was evaluated at the cellular level.The CCK-8 method was used to investigate the effect of the extraction on the viability of PC12 cells,and the changes in cell morphology,quantity,and fluorescence intensity between different treatment groups were observed through inverted microscopy and fluorescence microscopy.The results indicated that the extraction exerts its protective effect on PC12 cells by increasing the cell survival rate of H₂O₂-induced damaged cells,weakening the level of reactive oxygen species in damaged cells.The CCK-8 method was used to investigate the effect of extract from the leaves of Quercus dentata Thunb on the viability of A549,Huh7,and HCT116 tumor cell lines,and the morphological and quantitative changes of cells between different treatment groups were observed through inverted microscopy to preliminarily evaluate its inhibitory activity on tumor cell proliferation.The results showed that the morphology of A549,Huh7,and HCT116 cells in the treatment group with the extraction were damaged,and the number of cells were significantly reduced,indicating that the extraction had an inhibitory effect on these three cell activities.At the same concentration,the extraction had the most significant inhibitory effect on the activity of HCT116 cells.After 36 h treatment with the extraction,the IC₅₀ values of A549,Huh7 and HCT116 cells were 597.8.7±38.60μg·m L^-1,495.37±53.99μg·m L^-1 and 233.67±27.89μg·m L^-1,respectively.This will lay the foundation for further study of its anti-tumor mechanism.