Study On The Isolation,Structural Characterazation And Activity Of Phenolic Compounds From Adlay(Coix Lachryma-jobi L.var.ma-yuen Stapf)Seeds

Adlay is one of the oldest crops in China.Adlay,known as the "king of gramineous plants",is a kind of traditional healthy food and has high nutritional value.Studies on the active ingredients of adlay have always been the research focus in related fields.In this study,adlay seeds were first extracted with 80% acetone/water to obtain crude acetone extracts.Then the crude acetone extracts was fractionated with n-butanol to obtain n-butanol fraction.Furthermore,the n-butanol fraction was loaded onto a Sephadex LH-20 resin-packed column to yield three sub-fractions.Finally,sub-fractions were purified by semi-preparative HPLC to yield four compounds and the structures of these compounds were characterized by ESI-MS,1H NMR,and 13 C NMR techniques.The total phenolic contents of extracts,fractions and sub-fractions was determined using the Folin-Ciocalteu colorimetric method.The antioxidant activities of various extracts and compounds from adlay seeds based on the oxygen radical absorbance capacity(ORAC)assay,peroxyl radical scavenging capacity(PSC)assay and cellular antioxidant activity(CAA)assay.These extracts and compounds were carried out for their anti-tumor activities by MTT assay.Furthermore,the anti-tumor effects of adlay seeds on tumor cells apoptosis were explored by observing the cells under inverted microscope and Western Blot test.1.Isolation and structural characterazation of phenolic compounds from adlay seedsAdlay seeds were first extracted with 80% acetone/water to obtain crude acetone extracts.Then the crude acetone extracts was fractionated with n-butanol to obtain n-butanol fraction.Furthermore,the n-butanol fraction was loaded onto a Sephadex LH-20 resin-packed column to yield three sub-fractions.Finally,sub-fractions were purified by semi-preparative HPLC to yield four compounds.The structures of purified compounds were characterized by HPLC-DAD-ESI-MS,1H NMR,13 C NMR and UV-vis techniques.The contents of major individual phenolic compounds in the crude acetone extract,fractions and sub-fractions are measured by HPLC-DAD.Results showed that the most abundant phenolic compound in crude acetone extract,n-butanol fraction,aqueous fraction,sub-fraction 1 and sub-fraction 2 was N1,N5-di-[(E)-p-coumaroyl]-spermidine at 12.33 ± 0.53mg/g,16.01 ± 0.81mg/g,4.41± 0.02mg/g,2.02 ± 0.03mg/g and 106.41 ± 3.43mg/g respectively,whereas the predominant phenolic compound in sub-fraction 3 was ferulic acid at 62.90 ± 2.12mg/g.The four phenolic compounds were identified as N1,N5-di-[(E)-p-coumaroyl]-spermidine,p-coumaric acid,ferulic acid,and rutin,respectively.2.Total phenolic content and antioxidant activities of all extracts and compounds from adlay seedsThe total phenolic contents of extracts,fractions and sub-fractions was determined using the Folin-Ciocalteu colorimetric method.The antioxidant activities of various extracts and compounds from adlay seeds based on the oxygen radical absorbance capacity(ORAC)assay,peroxyl radical scavenging capacity(PSC)assay and cellular antioxidant activity(CAA)assay.The comparison of the crude acetone extract and fractions found that the n-butanol fraction exhibited the highest total phenolic content(43.83 ± 2.94 mg GAE/g DW)and antioxidant activities(ORAC values = 717.65 ± 33.88?mol TE/g,PSC values =185.59 ± 15.92?mol TE/g,CAA values = 41.38 ± 0.55?mol QE/g).Of the three sub-fractions obtained by Sephadex LH-20 chromatography,sub-fraction 3 possessed the highest antioxidant activity(ORAC values = 2500.76 ± 65.10 ?mol TE/g,PSC values =1454.45 ± 72.55?mol TE/g,CAA values = 153.57 ± 8.51?mol QE/g)and total phenolic content(183.42 ±3.27 mg GAE/g DW).Of the compounds in sub-fraction 2 and sub-fraction 3,p-coumaric acid exhibited the highest ORAC values(6975.22 ± 210.36?mol TE/g),ferulic acid exhibited the highest PSC values(2367.95 ± 111.21),and rutin exhibited the highest CAA values(1143.88 ± 80.98?mol QE/g).3.Anti-tumor activities of all extracts and phenolic compounds from adlay seedsVarious extracts and compounds from adlay seeds were carried out for their anti-tumor activities against Hep G-2,MCF-7 and Ca Co-2 cancer cell lines by MTT assay.Results showed that crude acetone extract,n-butanol fraction,sub-fraction 2,sub-fraction 3,N1,N5-di-[(E)-p-coumaroyl]-spermidine,p-coumaric acid,and ferulic acid had a inhibition of proliferation capability to all three types of cells in the dose range that cancer cells can grow.And there is a certain dose-effect relationship and time-effect.Hep G2 cells were the most sensitive to various extracts and compounds,while Ca Co-2 cells were the most insensitive.Of all the extracts and compounds from adlay seeds,N1,N5-di-[(E)-p-coumaroyl]-spermidine showed the highest level of antiproliferative activity with the IC50 values of 46.34±3.99?g/m L(Hep G-2),61.52±6.02?g/m L(MCF-7),92.69±5.63?g/m L(Ca Co-2),respectively.4.Effects of N1,N5-di-[(E)-p-coumaroyl]-spermidine on apoptotic signalingThe Hep G2 cell lines were treated with different concentrations of N1,N5-di-[(E)-p-coumaroyl]-spermidine for 48 h.Cells were examined under an inverted light microscope.After incubation with N1,N5-di-[(E)-p-coumaroyl]-spermidine,the cellular morphology of Hep G2 cells was severely distorted with reduced cell proliferation and some cells turned round in shape,indicating that N1,N5-di-[(E)-p-coumaroyl]-spermidine could dramatically alter the morphological characteristics of Hep G2 cells and made them to apoptosis in vitro.Effects of N1,N5-di-[(E)-p-coumaroyl]-spermidine on the expression of cytochrome c,caspase-9,caspase-3,Bcl-2,and Bax in Hep G2 cells were also analyzed by Western Blot test.The results showed that N1,N5-di-[(E)-p-coumaroyl]-spermidine significantly up-regulated the expression of cytochrome c,caspase-9,caspase-3,and Bax,while down-regulated that of Bcl-2 in Hep G2 cells.And the expression of each protein showed a significant dose-dependent relationship with the concentration of N1,N5-di-[(E)-p-coumaroyl]-spermidine.The results indicated that,N1,N5-di-[(E)-p-coumaroyl]-spermidine inhibited the proliferation of human Hep G2 liver cancer cells in vitro by participating in mitochondrial mediated cell apoptosis.