Studies On Separation, Purification And Pharmacological Functions Of Flavonoids From Celery (Apium. Graveolens L.)

Celery(Apium.graveolens L.) was introduced from Caucasia into China in about fifteenth century.Ancient medicinal herbs records and modern research have proved that Celery has a series of medicinal properties,such as reducing blood press,sedation, promoting digestion,diuresis and moistening lung.Researchers at home and abroad had studied on the metabolic process in body of interrelated compound from Celery in the 1970's.Study on separation,purification and pharmacology function of Celery flavonoids is becoming a hotspot at present.Utilization of Celery resources has been seriously restricted because of variety difference,lower produce purity,and intricate compounds and pharmacology function from Celery.Therefore,to study the content difference and characteristic of Celery flavonoids for various varieties;to establish technology and analytical method of extraction and purification of Celery flavonoids and to identify medicinal properties of Celery flavonoids will play very important role in theoretical and practical significance for Celery.The distribution of flavonoids from different Celery resources in Hunan was studied, and characteristics fingerprints of Apigenin,Apiin from Celery with chromatographic technique were created in this paper.The analysis methods were established for determination of Celery total flavonoid,Apigenin and Apiin.The ethanol-extraction, purification optimal technology and monomer preparation process of flavonoids compounds from Celery were studied.Three monomer compounds of Celery flavonoids by macroporous resin,polyamide resin,high speed counter current chromatogram,polydextran gel column chromatography technology were obtained.The chemical structures of monomer flavonoids compounds were identified by ¹H NMR,¹³C NMR,UV,IR and MS. By using cellular elements technology,biological activity and pharmacological functions of Celery flavonoids compounds on resistance liver cancer,leukemia,lowering lipid, anti-inflammatory were studied.These researches provide theoretical and technical supports for exploitation of the Celery resources and natural Celery flavonoids medicine. The experimental results are as follows:1 Creating analytical methods for Celery flavonoidsThe determination method of total flavonoids in Celery was established by aluminium nitrate-spectrophotometry.Regression equation of Rutin standard sample is:C=0.9967A+ 0.0178,R²=0.992。The relative standard deviation(RSD) of precision,chromogenic stability,repeatability,samples recovery ratio test was 0.34%,1.11%,2.38%,1.98%, respectively.The determination method of Apigenin in Celery was established by High Performance Liquid Chromatography(HPLC) systems equiped with UV detector and Kromasil C₁₈(4.6mm×150 mm,i.d.,5μm) chromatographic analysis column.The mobile phase was acetonitrile-watwe(35:65,v/v).The column temperature was set at 35℃and run on split mode with a constant flow of 1.0 mL/min.The detection wavelength was set at 270 nm.A sample in pouring volume was prepared by 10μL.The detection limit(DL) of Apigenin was 18 ng/mL.The relative standard deviation(RSD) of stability,precision, repeatability,samples recovery ratio was 0.93%,0.93%,1.08%,1.91%,respectively.The determination method for Apigenin in ethanol-extraction was established by High Performance Thin Layer Chromatography(HPTLC) using G60 silica gel plate (10cm×10cm).The developing solvent was chloroform-methanol-water(18:2.3:0.35, v/v/v).All analytical determinations were performed by deuterium lamp and tungsten lamp lighting,single wavelength reflexology absorption scanning in 345 nm,scanning speed 5 mm/s,scanning time 3 min,slit 5.00×0.30mm.The stability,precision and sample recovery ratio experiments were evaluated.The relative standard deviation(RSD) was 0.25%,1.33%,1.81%,respectively.The results show that the method was good stability in 30 min,high precision and high recovery ratio.The determination method of Apiin and 3'-methoxyl-Apiin in Celery was established by High Performance Liquid Chromatography(HPLC) for the first time.The systems fitted UV detector and ODSC₁₈(4.6mm×150mm,i.d.,5μm) chromatographic analysis column. The mobile phase was acetonitrile-watwe(23:77,v/v).The column temperature was set at 40℃and run on split mode with a constant flow of 0.6 mL/min.The detection wavelength was set at 270 nm.A sample in pouring volume was prepared by 10μL.The detection limits(DL) of Apiin,3'-methoxyl-Apiin was 42.00 ng/mL and 46.00 ng/mL,respectively. The relative standard deviations(RSD) of Apiin in stability,precision,repeatability and sample recovery ratio test was 1.4%,2.1%,3.1%,1.6%,respectively.The relative standard deviation(RSD) of 3'-methoxyl -Apiin in stability,precision,repeatability and sample recovery ratio test was 4.5%,2.1%,3.0%,0.8%,respectively. 2 Determinations of Celery flavonoids content and characteristics fingerprints for different Celery varieties in HunanThe contents of Celery flavonoids for seventeen Celery varieties in Hunan were measured by spectrophotometry,HPLC.The characteristics fingerprints of Apiin, 3'-methoxyl-Apiin were established.The contents of total flavonoids in dried Celery leaf in different production areas in Hunan were 1.541～20.350 mg/g,and the contents of flavonoids in dried celery leafstalk were 0.744～4.743 mg/g.the highest content of flavonoids was 20.350 mg/g in Zhangjiajie Wild Celery leaf,the lowest content of flavonoids was 1.541 mg/g in Shaoyang Solid Celery leaf.The contents of Apigenin in different local Celery varieties in Hunan were 0.003～0.088%.The contents of Apiin, 3'-methoxyl-Apiin in different local Celery leaf in Hunan were 0.12～0.43%,0.06～0.19%, respectively.The contents of Apiin,3'-methoxyl-Apiin in different local Celery leafstalk in Hunan were 0.01～0.04%,0.01～0.03%,respectively.The highest content of Apiin, 3'-methoxyl-Apiin in Zhangjiajie Solid Celery leaf was 0.43%,0.19%,respectively,and contents of Apiin,3'-methoxyl-Apiin were not detected in Zhangjiajie Wild Celery, Changsha Cress.The contents differences of flavonoids in different local Celery were large, and contents of flavonoids in celery leaf were higher than in in celery leafstalk.The content of Apigenin in Celery leaf increased with the increase of planting above sea level altitude, and decreased with the increase of planting above sea level altitude in celery leafstalk.3 Optimization of extraction process of Celery flavonoidsOn the basis of the single factor experiment,the extraction conditions of flavonoids in Celery(ethanol concentration,feed liquid rate,extraction temperature,extraction time) were optimized by orthogonal test.Optimal ethanol refluxing extraction condition of flavonoids from Celery leaf was for 70%ethanol extraction solvent;1:10(w/v) feed liquid ratio,90℃extraction temperature,and 120 min extraction time.The extraction ratio of Celery flavonoids can reach 70%in this condition.The ethanol extraction ratio of Celery leaf,leafstalk in different production areas in Hunan was measured;the results show that the ethanol extraction efficiency of different varieties was different because of the contents differences around local Celery varieties in Hunan.4 Creating techniques for Celery flavonoids Separation and purification and identifying chemical structures for new Celery flavonoids XDA-1 resin screened was applied to ennoblement of flavonoids from Celery extraction.Optimal conditions of dynamic adsorption and deabsorption was 2BV/h adding samples of flow velocity,eluting impurity by 8BV water lotion,eluting by 4BV of 70% ethanol and 2BV/h of flow rate.The dynamic adsorption and deabsorption ratio of XDA-1 resin was 88.18%,98.43%,respectively.The purity of flavone from Celery ethanol-extraction was increased to 10.52%,the ennoblement multiple was 8.416.The monomeric compound I was Apigenin(5,7,4'-Trihydroxyflavone) from Celery rude flavonoids via XDA-1 resin isolation,aether extraction and recrystallization after hydrolyzing 2 h with 1.2 mol/L hydrochloric acid at 80℃temperature.Its chemical structure was confirmed by ¹H NMR and ¹³C NMR,UV,IR,and MS analysis.Its purity was 98.0%by high performance liquid chromatography determination.The Celery rude flavone purified by XDA-1 resin was further isolated by polyamide column chromatography.Optimal separation conditions of polyamide column chromatography was 0.5 mL/min of adding samples flow velocity,eluting impurity by 3BV water lotion,eluting by 3BV of 40%ethanol and 1 mL/min of flow rate.The purity of Celery flavone was increased to 80%.A high-speed counter-current chromatography(HSCCC) method for the isolation and purification of flavone from the Celery crude extract was successfully established by using ethyl acetate -ethanol- acetic acid-water(4:1:0.2:5,v/v) as the two-phase solvent system at a flow rate of 2 mL/min and a revolution speed of 800 rpm at 25℃room temperature. Celery flavonoids glycosides monomer was tracked in 280 nm wavelength by HPLC, Celery flavonoids can very good separation.Their purity was 98.27%.The compoundâ…¡and compoundâ…¢from the Celery flavonoids purified by polyamide column chromatography were isolated by using Sephadex LH-20(1.6cm×80cm) column chromatography with 50%ethanol constant elution,tracking by HPLC.Their chemical structures were identified as Apigenin-7-(2-O-apiosylglucoside and Chrysoeiol-7-O-glucose -2-O-apiosyl glucoside by spectroscopic methods including ultraviolet,infrared, electrospay mass pectrometry and nuclear magnetic resonance(NMR).Their purity was 98.6%,98.0%,respectively,by high performance liquid chromatography determination. The two compounds were obtained from the Celery leaf for the first time. 5 Study on pharmacology activity of Celery flavonoids and monomer compoundsThe pharmacological activity of ethanol-extraction of Celery,Celery rude flavonoids, Apiin,3'-methoxy-Apniin,Apigenin were evaluated by using HTS models of FXR activation,FXR inhibition,Hypercholesterolemia cell strain(LXR),liver cancer cell strain (HepG2) and leukemia cell strain(HL-60).The results show Apigenin exhibited the highest inhibitory activity on LXR and increase the sensibility of FXR receptor at 10μg/mL of the samples concentration.Celery ethanol-extraction and Apigenin have inhibitory the effects at 10μg/mL the concentration for liver cancer cell multiplication and leukemia cell multiplication.GI₅₀ of ethanol-extraction of Celery is 66.07μg/mL,44.07μg/mL,GI₅₀ of Apigenin of Celery and Apigenin is 3.31μg/mL,1.62μg/mL,Apiin and 3'-methoxy-Apniin have not inhibitory effects for liver cancer cell and leukemia cell multiplication.Research results above would perfect theory and technology,and provide scientific proofs and methods for further processing of Celery.