A Preliminary Study On The Active Components And Mechanism Of The Anti-Inflammatory Of The Syringa Velutina Kom Leaves

ObjectiveTo study the different extraction technology of Syringa velutina Kom leaves.Compare the advantages and disadvantages of different extraction techniques and choose the best extraction technology of total flavonoids and total iridoid.To study the method of high performance liquid chromatography for the extraction of compounds in the Syringa velutina Kom leaves.To study the protective effect of Syringa velutina Kom leaves on acute liver injury in ICR mice induced by lipopolysaccharide and determine the effective site and main components.MethodsTwo phase solvents were used to separate the compounds.Using ethanol for cold soak,reflow,petroleum ether skim,total flavonoids were extracted from ethyl acetate and total iridoid were obtained from butanol.Using response surface method studied different factors effects.The effects of different extraction factors on the content of total flavonoids and total cycloenethers were compared by response surface method.The optimal combination of temperature,extraction time and solvent usage was investigated on the basis of single factor investigation.The content of total flavonoids extracted from ethyl acetate was determined by UV.High performance liquid chromatography(HPLC)was established to determine the content of Oleuropein,tyrosol,protocatechuicacid,astragalin,rutin and isoquercetin and the content of six substances in different harvest periods were compared to determine the best harvest time.The acute liver injury model of ICR mice induced by lipolysaccharide was established.The anti-inflammatory experiment was conducted by using the extractive of the Syringa velutina Kom leaves.The contents of ALT,AST,SOD,MDA,TNF-a and ICAM-1 in serum of acute liver injury were detected by ELISA method and the changes of hepatic pathological morphology of mice were observed under optical microscope by using HE staining and immunohisto-chemistry staining.The expression of inflammatory substances in the liver of mice were observed by using the immune imprinting technique.Results1、The content determination method of total flavonoids in ethyl acetate extracted and total iridoid in the n-butyl alcohol were established by using UV.Both of them in the extraction site were more than 50%.It was the effective extraction parts of the Syringa velutina Kom leaves.2、The response surface design is continuous and the accurate extraction scheme can be analyzed by fitting the data with the response surface model.The extraction rate was highest under optimal extraction methods.However,the influence factors and the interval need to be further expanded.3 、 Through the response surface method the total flavonoids and total iridoid quality are 9.754 g.under the condition,100 g the Syringa velutina Kom leaves has flavonoids extract quality is 4.741 g,the extraction rate is 2.784%,extract the determination of flavonoids content amounted to 58.72%;Total iridoid concrete quality is 5.013 g,the extraction rate is 3.166% content amounted to63.16%.4 、 HPLC was established to sure the contents of oleuropein,tyrosol,protocatechuic acid,Astragalin,rutin and isoquercetin in the Syringa velutinaKom leaves.Chromatographic conditions,the mobile phase was methanol(A)-0.6% phosphoric acid aqueous solution(B),gradient elution,detection wavelength was 250 nm,column temperature was 30 ℃.The flow rate was 1m L/min.The six ingredients were completely separated in 30 min.The regression equation and the linear range are in turn:Rutin Y=121447X-64136,13.8-137.6;Tyrosol Y= 116207x-24092,12.8-128.0;Protocatechuic acid Y=46915X+39201,9.9-99.2;Oleuropein Y=145157X-11907211.8-118.4;Astragalin Y=97109X-2359,10.6-105.6;Isoquercetin Y=236284X-9372.4,13.4-134.4.The average recovery rate was 99.04%(RSD = 1.09%),99.17%(RSD = 1.12%),98.83%(RSD = 0.74%),98.83%(RSD = 0.64%),99.00%(RSD= 0.75%),99.21%(RSD = 0.92%).5、Syringa velutina Kom leaves flavonoids and total iridoid extract can reduce the activities of ALT and AST in serum of mice caused by LPS,decrease MDA content,the influence degree is positively related to the dosage.The Syringa velutina Kom leaves flavonoids,total iridoid extract high and Low concentration can improve the liver of the SOD activity,compared with LPS group had significant difference,the water extraction of low dosage group had no significant difference.6、 Syringa velutina Kom leaves flavonoids and total iridoid extract can reduce the liver enlargement caused by inflammation,obviously decrease the LPS group liver index.7、Dyeing results:HE dyeing result showed that the flavonoids extract and total iridoid extract group,the liver cells damage,inflammatory overflow,cell membrane damage were reduced,compared with LPS.The difference has significant,and compared with the blank group,difference is not obviously.The results of immunohistochemistry staining showed that in the total iridoid extract and flavonoids extracts of experimental group tumor necrosis factor(TNF alpha)and inter-cellular adhesion molecule 1(ICAM 1)expression weresignificantly lower,the difference is statistically significant.Water extract group had no significant difference.The pathological examination results showed that;The iridoid and flavonoids extracted from the Syringa velutina Kom leaves can alleviate the pathological damage of liver cells and protect the liver injury model of acute liver injury.8、Western blot result:Total flavonoids and total iridoid extract of the Syringa velutina Kom leaves can inhibition expression of protein levels of TNFalpha and icam-1 induced by LPS,reduce the protein content of TNF-alpha and ICAM-1.Compared with LPS group,there was a significant difference and there was no significant difference compared with the control group.The water extract group inhibit the expression of protein levels of TNF-alpha and icam-1 induced by LPS.But the effect is not obvious.9 、 The total flavonoids extract and total iridoid extract of the Syringa velutina Kom leaves were able to improve the external physical and mental status of rats with acute liver injury,increased activity and feeding,and improved survival rate of mice.The anti-inflammatory effect were total iridoid>total flavonoids> water extraction.ConclusionsThe UV was easy to operate,easy to master and had reliable results.can used to determined the content of total flavonoids and total iridoid in the Syringa velutina Kom leaves.HPLC method can be used to determine the content of rutin,tyrosol,protocatechuic acid,astragalin,isoquercetin and oleuropein.This method had high sensitivity,good linearity,high recovery rate and accurate result.Determined that total flavonoids extract and total iridoid extract were effective parts of anti-inflammatory diseases.Extract can reduce the expression of inflammatory factors and inhibit the inflammatory lesions of hepatic cells.It can treat acute liver injury in mice and has protective effect on liver.