Studies On The Chemical Constituents,Content Determination And Lnhibitory Effect Of Tyrosinase Of Lignum Xanthocerais

Objective: Lignum Xanthocerais is the dry stem or branch of Xanthoceras sorbifolia Bunge.Wild in 52~2260 m elevation hills and other places,often cultivated in various places.Originated in Inner Mongolia in the north and Liaoning in the northeast of China.Xanthoceras sorbifolia Bunge is commonly used in Mongolian medicine.Mongolian medicine is named Sila Sendeng.Taste sweet,slightly bitter,can dispel rheumatism,relieve swelling and pain,mainly used for the treatment of rheumatoid arthritis,rheumatic internal heat,skin wind-heat and other symptoms.The chemical constituents of Xanthoceras sorbifolia Bunge mainly include triterpenoids,flavones,phenolic acids and sterides.Xanthoceras sorbifolia Bunge has many pharmacological activities such as anti-inflammation,anti-cancer,anti-oxidation,whitening,improving learning and memory function,and treating cardiovascular diseases.Therefore,this paper intends to systematically study the phytochemical constituents of Xanthoceras sorbifolia,on the basis of which the higher content components are enriched,and establish a method to determine the contents of six chemical constituents and the total flavonoids in Xanthoceras sorbifolia and investigate its extraction process.The effects of ethanol extract,extract fraction and 10 monomer compounds on the inhibition of tyrosinase were investigated.The aim is to enrich the chemical composition of Xanthoceras sorbifolia and provide an effective scientific basis for the quality control and development and utilization of Xanthoceras sorbifolia.Methods: By using a verity of chromatographic techniques,such as macroporous resin,HP-20 macroporous adsorption resin,Sephadex LH-20 column chromatography and ODS and chromatographic methods,24 monomer compounds were isolated from70% ethanol extract of Xanthoceras sorbifolia Bunge,and the structures of 17 compounds were identified by modern spectroscopy(UV,IR,MS,NMR,HMBC,HSQC,etc).In-depth and systematic phytochemistry research found that there are a large number of flavonoids in Xanthoceras sorbifolia Bunge.Therefore,myketin,a higher content component,was selected as the reference substance.In this study,a method for the determination of total flavonoids in Xanthoceras sorbifolia Bunge was established by ultraviolet spectrophotometry,and the total flavonoids in nine batches of Xanthoceras sorbifolia Bunge were determined.In this study,a method for the determination of(-)-epigallocatechin,catechin,epicatechin,dihydromyricetin,dihydroquercetin and myricetin in Xanthoceras sorbifolia Bunge by high performance liquid chromatography was established.The chromatographic conditions and extraction conditions were optimized.The chromatographic conditions and extraction technology were optimized,and the contents of the above six chemical constituents in 9 batches of Xanthoceras sorbifolia Bunge were determined.In this study,the inhibition rates of tyrosinase activity of naphthol,myricetin,quercetin,naringin,eriodictyol,dihydroquercetin,dihydromyricetin,dihydrokaempferol,5,7,3',4',5'-pentahydroxy flavanone and aescinated chemical components in Xanthoceras sorbifolia Bunge were determined by enzyme label analyzer.Results: Twenty-four compounds were isolated and purified from ethyl acetate and n-butanol fractions of the alcohol extract of Xanthoceras sorbifolia,including nine flavonoids,including kaempferol(1)?quercetin(2)?myketin(3)?naringenin(4)?eriodictyol(5)?dihydro-kaempferol(6)?dihydroquercetin(7)?dihydromyricetin(8)?5,7,3',4',5'-pentahydroxy flavanone(9),[(9S,10 R,11E,13R)-9,10,13-trihydroxyoctadec-11-enoic-acid)](10)?protocatechuic acid(11),(-)-epigallocatechin(13)?catechin(14),(-)-epicatechin-5-O-?-D-glucopyrnaoside(15),esculetin(12),?-sitosterol(16)?daucosterol(17).The content of total flavonoids in Xanthoceras sorbifolia was determined in this experiment.The results showed that the content of total flavonoids was 14.62-39.93 mg.g-1,and the contentsof six chemical components in Xanthoceras sorbifolia were also determined.The results showed that the contents of epigallocatechin,catechin,epicatechin were2.01-7.10 mg.g-1,0.11-1.37 mg.g-1,1.33-4.26 mg.g-1 and 1.06-5.42 mg.g-1respectively.-1.The contents of dihydroquercetin and myricetin are 0.03-0.95 mg/g-1and 0.05-1.40 mg/g-1 respectively.In this paper,tyrosinase inhibition method was used for the first time to investigate the activity inhibition rate of 10 monomer compounds in Xanthoceras sorbifolia.The results showed that except dihydroquercetin and sage grass phenol,all the components tested had the effect of inhibiting tyrosinase activity.Conclusion: Compounds 1,4,9 and 11 ~12,15 were isolated from Xanthoceras sorbifolia Bunge for the first time.Compounds 5~6,10 were isolated from this genus for the first time.A method for the determination of total flavonoids in Xanthoceras sorbifolia Bunge by ultraviolet spectrophotometry and a method for the determination of six chemical constituents in Xanthoceras sorbifolia Bunge by high performance liquid chromatography were established.The two methods are simple,accurate and reproducible,and can be used for the quantitative study of Xanthoceras sorbifolia Bunge flavonoids.This study found that most flavonoids have tyrosinase inhibitory effect,which laid a foundation for further study of the effect of flavonoids on tyrosinase activity.