Study On The Extraction, Isolation And Bioactivities Of Flavonoids In Dalbergia Odorifera T. Chen Seeds

Dalbergia odorifera T. Chen belongs to Dalbergia of Leguminosae. The dried heartwood orroot of the plant namely Jiangxiang is a well-known and valuable Chinese herb and has beenused as a main ingredient in many formulae to treat cardiovascular diseases. Flavonoids andvolatile oil are major components in Jiangxiang. The plant needs several decades to grow untilit can be used while satisfactory yield of seeds can be obtained after5years. However, upuntil now, very few data on chemical composition and bioactivity of flavonoids in seeds of theplant are available. The aim of the present study was explored the determination, extraction,isolation, identification, complexation modification and bioactivities of flavonoids in theseeds and provided a scientific support for the utilization of them.Using rutin as standard, NaNO₂-Al（NO3）3-NaOH as color developing reagent, the contentof flavonoids in seeds of the plant was determined by spectrophotometry. Results showed thatthe accuracy, stability and recovery of the method were adequate and the content offlavonoids in seeds of the plant was3.83%. The reflux extraction of flavonoids in seeds of theplant was investigated by single factor experiments. The optimum extraction conditions wereas follows:50%ethanol as solvent, ratio of solid to liquid1:20, extraction at70℃for1h and2times. At the condition, the extraction rate and yield of flavonoids were91.70%and3.51%,respectively.In vitro antioxidant activity of the50%ethanol seed extract of the plant and its fourextractive fractions was investigated by six different methods. The ethyl acetate fractionexhibited the highest total phenolics （563.2mg GAE/g）, total flavonoids （350mg RE/g）,reducing power (IC₅₀=0.36mg/mL), DPPH radical scavenging activity (IC₅₀=0.17mg/mL)and activity in preventing the peroxidation of linoleic acid （64.4%）. The water fractionshowed the best ferrous chelating ability (IC₅₀<0.5mg/mL). Positive linear correlations wereobserved between above methods except ferrous chelating ability.In addition, hypoglycemic activity of above five extracts was evaluated by two methods.All extracts showed weak inhibitory effects on α-amylase. However, the petroleum etherfraction showed much higher α-glucosidase inhibitory activity than the standard reference ofacarbose, and the ethyl acetate fraction showed lower α-glucosidase inhibitory activity thanacarbose but still exhibited good potential to be used as a new product for the treatment ofdiabetes.LC-MS analysis of the ethyl acetate fraction was performed and results showed that therewere four main compounds at280nm and named compoundⅠ-Ⅳ, respectively. CompoundⅠand Ⅱ were isolated by silica column chromatography and crystallization and identified bymeans of IR, LC-MS,1H and13C. Compound Ⅰ, a new isoflavone, was identified as 5,7-dihydroxy-2’,3’,4’,5’-tetramethoxy isofavone. Compound Ⅱwas identified astectorigenin. The two pure compounds showed lower DPPH radical scavenging activity thanthe ethyl acetate fraction. Compound Ⅰand the ethyl acetate fraction had similar inhibitoryeffects on α-glucosidase, as well as compound Ⅱ and acarbose. Both of them werenoncompetitive inhibitors of α-glucosidase.Copper, ferrum and zinc complexes of compound Ⅰ were prepared. All complexesshowed lower DPPH radical scavenging activity but higher α-glucosidase inhibitory effectsthan compound Ⅰ. The copper complex showed the highest inhibitory effect which was88.03%at the concentration of10μg/mL. By means of IR, TG and elemental analysis, the chemicalformula of copper complex was concluded as C19H17O8Cu.2H2O and the chelating sites werecarbonyl and5-hydroxy.