| Objective:In this article,phenolic acid from different tissues of Walnut were used as the research object,and the following works have been done:the evaluation of the antioxidant activity in vitro and vivo of phenolic acid from different tissues of Walnut,the determine of the content of 4 kinds of phenolic acids in the Diaphragma juglandis Fructus,the purification of total phenolic acid of Diaphragma juglandis Fructus and the evaluation of the antioxidant activity of different extraction parts and purified product of macroporous resin.Methods:1.The JugLans regia L.were used as the research object to optimized the extraction process of phenolic acids,the effect of the extraction solvent,extracting temperature,extracting time,solid-liquid ratio were studied by the single-factor experiment and orthogonal design using the extract rate of phenolic acids as index,and determined the content of total phenolic acids in different tissues of Walnut.2.The antioxidant activity in vitro of phenolic acids in different tissues of Walnut was evaluated by the determination of the reducibility,the capacity of removing DPPH·,hydroxyl radicals?·OH?,superoxide anion?O2-·?,H2O2 radicals and the spontaneous lipid peroxidation in rat liver homogenization.3.Using the Kun-ming mice as subjects,gavaging administration for four weeks,and the changes of body weight,organ index and the content of Superoxide Dismutase?SOD?and malondialdehyde?Malondialdehyde,MDA?in serum and liver were observed to evaluate the antioxidant capacity in vivo of phenolic acids in different tissues of Walnut.4.The HPLC analysis was performed on a Waters XSELECETTMGSHTMC18?250mm×4.6 mm,5?m?,the detection wavelength was 214 nm,temperature was 30?,flow rate was1.0 mL·min-1,the acetonitrile-0.1%H3PO4 as the mobile phase,and Agilent 1260 Infinity?high performance liquid chromatograph were used to establish the chromatogram to establish the method of determine the content of gallic acid,protocatechuic acid,methyl gallate and caffeic acid in the Diaphragma juglandis Fructus from different sources5.In this part,solvent extraction and macroporous resin were used in purification of phenolic acid total extract of Diaphragma juglandis Fructus;the single-factor and orthogonal design experiment were used to optimize the purification technolgy of macroporous resin HPD100,and compared the content of phenolic acid in different extraction parts and purified product of macroporous resin.6.Evaluated the antioxidant activity in vitro of purified products of solvent extraction and macroporous resin by the determination of the reducibility,the capacity of removing DPPH·,hydroxyl radicals?·OH?,superoxide anion?O2-·?,H2O2 radicals and the spontaneous lipid peroxidation in rat liver homogenate.7.Using Kun-ming mice as the subjects,gavaging administration for four weeks,and the changes of body weight,organ index and the content of SOD and MDA in serum and liver were observed to assess the antioxidant capacity in vivo of different extraction parts and purified product of macroporous resin.Result:1.The optimum conditions were ethanol concentration 40%,extraction temperature80?,extraction time 60 min,solid-liquid ratio 1:21(g·mL-1).The order of contents of phenolic acids in different tissues of Walnut as following:Walnut Kernel Pellicle>Diaphragma juglandis Fructus>walnut husk>Walnut leaves>Walnut shell>Walnut pollen>Walnut branch>flower of Juglans regia>Walnut kernel.2.The antioxidant capacity in vitro was judged by IC500 value,and the order of removing DPPH·of different tissues of Walnut and VitC as followings:Walnut kernel pellicle>Diaphragma juglandis Fructus>Walnut leaves>Vit C>Walnut shell>Walnut branch>Walnut husk>flower of Juglans regia>Walnut pollen>Walnut kernel,the reducibility order:BHT>Walnut kernel pellicle>Diaphragma juglandis Fructus>Walnut leaves>Walnut branch>Walnut shell>Walnut husk>flower of Juglans regia>Walnut pollen>Walnut kernel;The inhibition order of spontaneous lipid peroxidation in rat liver homogenate:VitC>Walnut kernel pellicle>Diaphragma juglandis Fructus>Walnut branch>Walnut kernel>Walnut pollen>Walnut leaves>Walnut shell>Walnut husk>flower of Juglans regia;Removing superoxide anion?O2-·?order:Vit C>Walnut kernel pellicle>Diaphragma juglandis Fructus>Walnut leaves>Walnut husk>Walnut shell>Walnut branch>Walnut pollen>flower of Juglans regia>Walnut kernel;Removing H2O2 order:VitC>Diaphragma juglandis Fructus>Walnut kernel pellicle>Walnut kernel>Walnut husk>Walnut leaves>Walnut pollen>Walnut branch>Walnut shell>flower of Juglans regia;Removing hydroxyl radicals?·OH?order:VitC>Walnut kernel>Walnut branch>Diaphragma juglandis Fructus>Walnut pollen>Walnut kernel pellicle>Walnut leaves>Walnut husk>Walnut shell>flower of Juglans regia.3.The determination results of SOD activity in serum of mice as following:Diaphragma juglandis Fructus>Walnut kernel pellicle>control group>Walnut pollen>Walnut shell>Walnut branch>flower of Juglans regia>Walnut husk>Walnut kernel>Walnut leaves>blank group;The order of SOD activity in mice was:control group>Diaphragma juglandis Fructus>Walnut kernel pellicle>Walnut shell>Walnut pollen>Walnut husk>Walnut branch>flower of Juglans regia>Walnut leaves>blank group;The order of MDA concent in the serum was:control group<Diaphragma juglandis Fructus<Walnut husk<Walnut kernel pellicle<flower of Juglans regia<Walnut shell<Walnut pollen<Walnut branch<Walnut kernel<Walnut leaves<blank group;The order of MDA concent in liver of mice was:Diaphragma juglandis Fructus<control group<Walnut kernel pellicle<Walnut shell<flower of Juglans regia<Walnut husk<Walnut branch<Walnut leaves<Walnut kernel<Walnut pollen<blank group.4.The average content of gallic acid,protocatechuic acid,methyl gallate and caffeic acid in the Diaphragma juglandis Fructus from different sources was 1.157 9 mg·g-1?0.087 09mg·g-1?0.050 96 mg·g-1?0.179 4 mg·g-1;and the order of the average content of the five components as followings:gallic acid>caffeic acid>methyl gallate>protocatechuic acid.5.The optimal adsorption of macroporous resin HPD100 in purification of phenolic acid total extract of Diaphragma juglandis Fructus as followings:the concentration in the solution of 5 mg·mL-1,the feed volume was 3 BV,the adsorption time was 8 h,and the feed flow rate was 1 mL·min-1.And then desorpted 10 h with 2 BV of absolute ethanol at flow rate of 1mL·min-1.6.The reducibility of different extraction parts and purified product of macroporous resin and BHT are as followings:BHT>Mrp>Eap>Nbp>TE;The scavenging ability was evaluated by IC500 value,the order of removing DPPH·as followings:Vit C>Eap>TE>Mrp>Nbp;the inhibition order of spontaneous lipid peroxidation in rat liver homogenate:VitC>Eap>Nbp>Mrp>TE;the order of removing O2-·:VitC>Nbp>TE>Eap>Mrp,and removing·OH and H2O2 order as followings:Vit C>Mrp>Nbp>Eap>TE.7.The determination results of SOD activity in serum and liver tissue of mice were as following:control group>Mrp>Eap>TE>Nbp>blank group;The order of MDA in the serum of mice was:control group<TE<Mrp<Eap<Nbp<blank group;The order of MDA content in serum liver of mice was:control group<Mrp<Nbp<Eap<TE<blank group.Conclusion:1.Based on the single-factor experiment and orthogonal design,optimized the extraction process of phenolic acids from walnut shell,the extraction process of total phenolic acids from walnut shell was optimized.The method is simple and quick,and the optimized process is practicable.2.The different tissues of Walnut is full of antioxidant activity in vitro,and all are linked to their concentration.3.According to the antioxidant experiments in vivo and in vitro,the result shows tha the antioxidant capacity of Walnut Kernel Pellicle and Diaphragma juglandis Fructus are better than others.4.The method of determine the content of gallic acid,protocatechuic acid,methyl gallate and caffeic acid in the Diaphragma juglandis Fructus has been established,and the method is simple and stabilize.Which is compliant for the determination of 4 components in the Diaphragma juglandis Fructus.5.The order of content of phenolic acid and the total content of 4 kinds in the purified products of solvent extraction and macroporous resin were as following:Mrp>Eap>Nbp>TE>Rwp,And the purification effect of macroporous resin is better than solvent extraction.6.The different extraction parts and purified product of macroporous resin is full of antioxidant activity in vitro,all were related to their concentration,and the antioxidant capacity of Eap and Mrp were better.7.According to the antioxidant experiments in vivo and in vitro,the result shows that the antioxidant capacity of Mrp was the strongest. |
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