The Preparation And Assessment Of Protection Against Oxidative Stress Of The Active Ingredient From Syringa Oblate Lindl.

Syringa oblata Lindl.,being a most widely landscape greenery,is distributed in southwest,north and northeast China.It has been extensively used as folk medicine for treating diarrhea and acute icteric hepatitis in China.Syringin and oleuropein are main effective components of early lilac,waiting for further development and utilization.Moreover,as a naturally active ingredient from plant,hydroxytyrosol?HT?have powerful antioxidation.It is mainly from Olive Mill Wastewater?OMWW?or juice of olive fruit and is used in many fields,such as food,cosmatic,drug,health care products.But the limited resource and separation technology are still difficult topics.So,in the present study,extraction and separation of syringin and oleuropein from Early lilac?Syringa oblata Lindl.?were investigated.And separated oleuropein was converted into HT using ion exchange resin.The results were as follows:1.The analysis method for simultaneous determination of syringin and oleuropein from Early lilac and an efficient vacuum microwave-assisted extraction?VMAE?were developed.?1?Reversed-phase chromatography was performed on a Hypersil BDS C18 column?4.6 mm ×250 nm,5 ?m?for the determination of syringin and oleuropein.RP-HPLC analysis of syringin and oleuropein was done according to the procedure as follows.The mobile phase was consisted of methanol:0.2%phosphoric acid in water?33:67,v/v?.The column temperature was maintained at 25 ?.The wavelength used for syringin and oleuropein with the diode detector was 232 nm.The flow rate was 1.0 mL/min and the injection volume was 10 ?L.This analysis method is accurate,precise,sensitive and raliable.An analytical method was developed on the determination of syringin and oleuropein for the further utilization of Early lilac.?2?An efficient vacuum microwave-assisted extraction?VMAE?was successfully applied to the extraction of syringin and oleuropein from early lilac.A study for optimizing the reaction conditons have been performed by Box-Benhnken design?BBD?based on the results of single-factor experiments.The optimum extraction process was as follows:extraction time,7 min;ratio of liquid/solid,20 mL/g;extraction solvent,ethanol-water?42:58,v/v?solution;microwave power,120 W;degree of vacum,-0.08 MPa;soaking time,1 h.Under optimal conditions the maximum yield of syringin and oleuropein were 5.16 mg/g and 3.52 mg/g,respectively.VMAE were timesaver and higher efficiency,especially for the extraction of thermosensitive glycosides,compared with the conventional method.2.The optimum process for the purification of syringin and oleuropein from Early lilac branchlet was obtained.?1?Separation and enrichment of syringin and oleuropein by macroporous adsorption resins was studied with the material of 40%ethanol extract of Early lilac branchlet.The seperation effects on HPD-100B resin was the best among 11 types of resins.?2?The optimum adsorption parameters on the HPD-100B resin were confirmed as follows:435 ?g/mL and 422 ?g/mL for concentration of syringin and oleuropein in extraction solution;Adsorption temperature was 25 ?.The flow rate was 3 BV/h.The adsorption capacitys were 12.2 mg/g and 51.5 mg/g resin for syringin and oleuropein,respectively.Moreover,the effluent from resin column among 15BV-60BV contains a great quantity syringin with few impurities.This part contains syringin 34.4 mg/g resin.?3?The optimum desorption parameters on the HPD-100B resin were confirmed as follows:20%ethanol 7 BV was selected to wash syringin and 40%ethanol 9 BV to elute oleuropein.The flow rate was 3 BV/h.After HPD-100B resin column adsorption,the content of syringin increased to 7.2%,increased 7.1 times;the content of oleuropein increased to 14.2%,increased 8.2 times.The recoveries of syringin and oleuropein were 77.4%and 89.8%,separately.3.The further purification of syringin and oleuropein was done from the products of macroporous adsorption resin column with silica gel column chromatography.The optimum conditions of silica gel column chromatography for syringin and oleuropein were confirmed as follows:?1?The further purification of syringin was done as follow parameters:the quantity ratio of sample/ODS silica gel was 1:40;the flow phase was EA-MeOH-H2O?12:1:0.2,v/v/v?;the mesh of silica gel was 100-200;the flow rate was 0.6cm/min.And after this process the content of syringin was 92%.The recovery was 78%??2?The further purification of oleuropein was done as follow parameters:the quantity ratio of sample/ODS silica gel was 1:70;the flow phase was EA-MeOH-H2O?16:1:0.2,v/v/v?;the mesh of silica gel was 100-200;the flow rate was 0.6 cm/min.And after this process the content of syringin was 86%.The recovery was 85%?4.An efficient catalytic hydrolysis technique was developed to prepare HT from oleuropein with an ion exchange resins as a heterogeneous catalyst.?1?The performances of eight types of anion exchange resins have been evaluated.The research results indicated that 001*7,D72,D61 resins were better than the others.The most appropriate anion exchange resins for convention of oleuropein into HT was 001*7.?2?The optimum parameters on the anion exchange resins were confirmed by static hydrolysis as follows:hydrolysis and desorption temperature was both 70 ?;the ratio of catalyst amount/volume of oleuropein solution was 4%?w/v?;hydrolysis time was 2 h;desorption time was 1h;the concentration of oleuropein solution was 574.2 ?g/mL;eluting agent was 80%ethanol.?3?After the hydrolysis of 001*7 resins column,hydrolysis rate of oleuropein was 96.37%,which was higher than the static hydrolysis's 81.6%.An efficient catalytic hydrolysis technique was developed to prepare HT from oleuropein with an ion exchange resins as a heterogeneous catalyst.The ion exchange resins were reusable.This technique has mild reaction condition,convenient disposition,good yield,safety and so on.In addition,it avoid the problems that existing in hydrochloric acid hydrolysis such as acid pollution and difficult separation.5.Assessment of HT and oleuropein on protection LLC-PK,cells in oxidative stress induced by H₂O₂.?1?Oxidative stress damage cell model could be established in LLC-PK,cells induced by 100 ?m H₂O₂ for 4 h.?2?HT?2 ?M?and Oleuropein?10 ?M?could effectively reduce MDA content and SOD,GSH-Px activities in cells,thereby preventing the damage to renal cell caused by free radicals.?3?Composition of fatty acid of H₂O₂ model group were changed compared with blank control group.The relative contents of unsaturated fatty acids were significantly reduced.HT and Oleuropein could effectively inhibit transformation of saturated fatty acids of LLC-PK1 cells treated with H₂O₂.HT and Oleuropein had inhibitory activity on LLC-PK1 cells in oxidative stress induced by H₂O₂.And HT has been shown superior to Oleuropein.