Research Of The Impacts Of Lygodium Japonicum(Thumb.)Sw On Angiogenesis In Chick Embryo Chorioallantoic Membrane And The Preparation Of The Effective Composition

Lygodium japonicum(Thunb)Sw.,which is Herbaceous perennial vine,belongs to the family of Lygodiaceae and the genus of Lygodium.Studies have shown that Lygodium has detoxification,gallbladder,swelling,antibacterial pharmacological activities.However,the antiangiogenic effects of Lygodium have been reported rarely.In this paper,chicken chorioallantoic membrane(CAM)model was used in the anti-angiogenic activity of site selection of Lygodium,and then make the active site of extraction,refining.In addition,analyse the blood components by adopting serum pharmacochemistry and HPLC analysis method.To provide certain experiment basis for revelation the effect material of anti-angiogenesis in Lygodium.Objective:In this paper,in order to obtain an antiangiogenic active site from Lygodium extracts,CAM model was used to filter.Then identify,extract and refine it.To provide experimental evidence for the development and application of the Lyodium.Methods:1.Adopt system separation,successively using petroleum ether(PE),ethyl acetate(EA)and butyl alcohol(BA)as the extraction solvent,to extract the extracting solution from dried whole plant Lygodium.Prepare PE extract,EA extract and BA extract.2.Adopt chicken chorioallantoic membrane(CAM)model,the neovascular growth percentage in dosing mouth was taken as the index.Compare the anti-angiogenic activity of these three kinds of extracts.3.Using color reaction,hydrochloric acid-magnesium,hydrochloride-zinc,complex reaction of aluminum chloride and 4%sodium hydroxide solution reaction and UV-visible spectroscopy and through establishing the fingerprint to analysis the active site of againsting angiogenesis,confirm the ingredients categories of Lygodium BA extract.4.Prepare active site of anti-angiogenesis.Use chick embryo chorioallantoic membrane(CAM)model to verify and employed chick embryo yolk sac membrane(YSM)model for further study.5.Using L9(34)orthogonal method,the yield rate and the contents of angiogenic activity site as targets,screened Lygodium Extraction process.Investigated the effect of solvent concentration,dosage,extraction times on the extraction results to determine the optimal extraction conditions.Using the method of refrigerated standing,the active ingredient content and chlorophyll clearance rate were as the targets.Study the effects of concentrations of the extract removing the chlorophyll.6.Using column separation technology to separate extracts from active site,then get different concentration of ethanol elution parts,with CAM model for screening of anti-angiogenesis effect and conduct validation test.7.Adopted serum pharmacochemistry,Use 50%ethanol elution parts as drug group for lavage treatment,prepare blank serum group?Blank serum + drugs group?drugs group and drug-containing serum group,then use the method of HPLC analysis to Analyse the blood components.Result:1.Lygodium PE extract,EA extract and BA extract have a certain extent on chick chorioallantoic membrane vessel growth,the BA extract expresses the strongest inhibition,followed by the EA extract.2.Use hydrochloric acid-magnesium and hydrochloride-zinc reaction tests for detection of Lygodium BA extract,the result is red foam;it is yellow when using complexation reaction test of aluminum chloride;while it is brown-red color reaction when using 4%sodium hydroxide solution for detection.UV-visible spectroscopy shows that the absorption peaks of Lygodium BA extract and rutin were nearby at 510nm after colorate.By establish the HPLC fingerprint of and analyze the absorption spectrum characteristics.Based on the above results,Preliminary judgment is that Lygodium BA extract contains mainly about flavonoids.3.The angiogenesis significantly reduced when high and middle dosage drug worked on CAM model,the density of angiogenesis growth rate and the number were significantly reduced when high and middle dosage drug worked on YSM model,it can speculated that the total flavonoids in Lygodium has a certain inhibition on vascular,and in a dose-dependent manner.4.Using L9(34)orthogonal method to screen process of extraction,it is known by range analysis and variance analysis results,that the impact on the effective ingredient extraction rate factors are the order of the ethanol concentration>extraction time>solvent consumption,and the results show that the concentration of ethanol have significant differences,The best technology to determine the extracte conditions of total flavonoids from Lygodium is:ethanol concentration of 50%,the ethanol dosage as 18 times the amount of medicine,extracted 2 times and 2h per time.5.Use the method of refrigerated standing to remove the chlorophyll from the extract.The results suggested that the chlorophyll content is the lowest when the crude drug concentration is 0.5g/ml.6.After column separation,we get four kinds of sites eluted by different concentrations of ethanol.The site eluted by ethanol in concentration of 50%show that it is the strongest inhibition of chick angiogenesis.Determine the site as the effective part of the anti-angiogenesis in chick embryo.Concentration of 9.6g crude drug/ml is determined as the optimal.7.Adopt serum pharmacochemistry and using the methord of HPLC analysis to analyze of serum in OVX.By comparing each chromatogram,There are six components retain the same time and five new absorption peaks.Probably after absorption,the five components are metabolite,It needs further study to determine the components.Conclusion:This study is a combination of Lygodium progressively separated form and chicken chorioallantoic membrane(CAM)model.The best site to inhibit angiogenesis in chick embryo is the site that eluted from flavonoids by concentration of 50%ethanol.Using L9(34)orthogonal method and the method of refrigerated standing,it could summed up that the extracte conditions of total flavonoids from Lygodium are:ethanol concentration of 50%,the ethanol dosage as 18 times the amount,extracted 2 times and 2h per time.Keep the crude drug concentration as 0.5g/ml when using the method of refrigerated stangding to clear chlorophyll,and adopting serum pharmacochemistry,the mice are treated with the site.Analyze the serum by HPLC.There are six components retain the same time and five new absorption peaks.Probably after absorption,the five components are metabolite,It needs further study to determine the components.