Anti-Oxidative Constituents of Ethanol Extract from Buckwheat Seeds by HPLC-Electro-Spray MS

Agricultural Sciences in China2008,7(3):356-362° Science directMarch 2008Anti-Oxidative Constituents of Ethanol Extract from Buckwheat Seeds bHPLC-Electro-Spray MsYAO Ya-ping, TIAN Cheng-ruiI and CAo Wei2I College of Food Engineering and Nutrition Sciences, Shaanxi Normal University, Xi' an 710062, P R China2 College of Chemical Engineering, Northwest University, Xi'an 710069, P R ChinaAbstractThe study investigates major anti-oxidative constituents of ethanol extracts from the seeds of common buckwheat andtartary buckwheat. Ethanol extracts from buckwheat seeds were arranged to react with 1, 1-diphenyl-2-picrylhydrazyl(DPPH)free radical. HPLC was used to identify anti-oxidative constituents of the ethanol extracts, and electro-spray Mswas used to characterize the structures of these identified anti-oxidative constituents to confirm them. the ethanolextracts of common buckwheat and tartary buckwheat seeds both had DPPH free radical-scavenging effect; HPLC analysisshowed that the ethanol extracts of both common buckwheat and tartary buckwheat seeds presented two main antioxidation peaks, which correspondingly had same chromatographic retention times and spectral information; electro-spray MS analysis showed that the molecular weights and Ms fragmentation patterns of the anti-oxidative constituentsin the ethanol extracts from buckwheat seeds were the same as those of rutin and quercetin in the control samples. HPlCMS/MS was capable of being used to rapidly identify anti-oxidative constituents in the extract of buckwheat seeds, andhe main anti-oxidative constituents of buckwheat seed extract were mainly rutin and quercetin, and the anti-oxidativeactivity of quercetin was higher than that of rutin.Key words: buckwheat, anti-oxidative constituent, HPLC, MS/MSsubstances for a long term( florian et al. 2007; MachlinINTRODUCTION1995). Because people are afraid that chemicallysynthesized anti-oxidants may have certain harmfulResearches in free radical biology show that the incidence effects, it is of great importance to identify natural antiand progress of human diseases, such as cancer, aging, oxidative constituents in daily food.and cerebrovascular, and cardiovascular diseases are Buckwheat, a plant belonging to fagopyrum ofrelated to free radical excess in human bodies(Kaliora polygonaceae, is widely planted in China and many otheret aL. 2007; Valko et aL. 2007). Free radicals are capable countries of the world and its commonly planted speciesof not only injuring tissues and organs but also destroying are common buckwheat and tartary buckwheatcellular components, thereby affecting normal cellular (Jayaprakasha et aL. 2007). Recent researches(Linfunctions and resulting in disease incidence et al. 2002: Oomah and Mazza 1996: Watanabe 1998)(Ramarathnam et al. 1995). Epidemiology researches have found that buckwheat seed extract has a stronghave found that it can effectively reduce the incidences anti-oxidative activity, which have something to do withof cerebrovascular and cardiovascular diseases, and the high contents of its anti-oxidative constituents. Forcancers to take a certain amount of anti-oxidative investi中国煤化工 dative constituentsCNMHGThis paper is translated from its Chinese version in Scientia Agricultura Sinica.YAOYa-ping.PhD,Tel:+86-29-85224397.E-mail:yaoyaping2005@126.com;CorrespondenceTIANCheng-rui,Tel:+86-29-85310518,E-mail:chian@snnueducno2008, CAS. A ights reserved Pubished by EbeviorLH.Anti-Oxidative Constituents of Ethanol Extract from Buckwheat Seeds by HPLC-Electro-Spray MSof buckwheat, differently polar organic solvents are used 5 mL 70% ethanol to carry out the extraction of itsin most cases to extract the anti-oxidative constituents anti-oxidative constituents at room temperature for 24and then their anti-oxidative activities were measured. h without direct lighting. The solution of the extractionThe main disadvantage of this method is that it cannot was filtered through a filter paper and then the residueprovide any structural information of the anti-oxidative of the extraction was mixed with 5 mL 70% ethanol toconstituents in the extract( Quettier-Deleu et al. 2000). carry out one more extraction under the same conditionsHPLC-electro-spray ion trap MS(HPLC-EIT-MS) for 24 h. The solutions from the two extractions weretegrates the separation by chromatography and the mixed and the mixture was centrifuged at 8000 r/minualitative determination by MS into one thus capable for 10 min; the supernatant of the mixture wasof providing structural information of test substances condensed at 40 C by decompression and its ethanolin a complex system without any preparatory treatments, was retrieved at the same time. Next, the residual solutionand as a result it has obvious advantages in investigating of the supernatant was lyophilized to form the ethanolanti-oxidative constituents in any complex systems(Sun extract of buckwheat seed flour; then 10 mg of theand Ho 2005; Shui and Peng 2004 Toshimasa et aL. ethanol extract was taken by weighing and was2003; Koleva et al. 2000). In this study, HPLC-ElT- dissolved in 5 mL ultra-pure methanol, and the solutionMS was adopted to investigate and identify the main was filtered with 0.45 um micro-porous film. Theanti-oxidative constituents of buckwheat.filtrate was used for anti-oxidation examination andHPLC-EIT-MS analysisMATERIALS AND METHODSExamination about dPPh free radical-scavenging effects of the ethanol extract ofInstruments and reagentsbuckwheat seedsAgilent 1200 series rapid resolution LC system(Aglient For the examination, the method developed by LarraunTechnologies, USA); diode array detector; Zorbax SB- et al. 1998)was adopted, but some modifications wereC18 column(150 mm x 4.6 mm, 5.0 um); UV-VIs made to it. Different volumes of the ethanol extract ofspectrophotometer( Beijing Purkinje General Instrument buckwheat seeds were taken and separately dissolvedCo, Ltd.in 4 mL methanol solution with DPPH at 0.025 mg mL;1, 1-diphenyl-2-picrylhydrazyl (DPPH)purchased and then the mixed solutions were resized to 5 mLm Sigma of America; rutin and quercetin(as theAfter the mixed solutions had been kept still at roomcontrols)purchased from the National Institute for the temperature for I h without direct lighting, their opticalControl of Pharmaceutical and Biological Products of densities were measured at 516 nmChina; chromatographic pure methanol; ultra-pure The DPPH free radical scavenging capacity of awater;and analytical pure phosphoric acid and ethanol. sample could be calculated by the following formulaDPPH free radical scavenging capacity =[1Samples and sample preparationDPPHDPPH×100%Where, DPPH,m stands for the optical density of aIn this study there were two buckwheat varieties sample at 517 nm and DPPHnm stands for the opticalinvolved, Qianwei 3( tartary buckwheat)and Pingqiao density of the control at 517 nm.3 (common buckwheat), and the seeds were providedby the Guyuan Research Institute of Agricultural HPLC identification of major anti-oxidation peaksSciences and Technology of Ningxia, China.that the ethanol extracts of buckwheat seedsThe buckwheat seeds were ground into flour using presa plant sample pulverizer and then the flour was sieved中国煤化工through a 60-mesh screen. Next, 1 g of the buckwheat 1 mlCN MH Gkwheat seeds wasflour was accurately taken by weighing and mixed with added to 1 mL 2.5 mmol L: ' DPPH solution and wasC2008, CMS. AI rght reserved Publshed by BawviwrLYAO Ya-ping et aL.kept to react at 37 C for 1 h to form the sample solution. of the ethanol extracts of buckwheat seeds at differThe control solution was prepared with the ethanol concentrations. According to Fig. 1, whileextract of buckwheat seeds and methanol. The sample concentrations of the ethanol extracts of buckwheatsolution and control solution were filtered with 0. 45 seeds increased, the DPPh free radical scavengingum micro-porous film and their filtrates were directly capacities correspondingly gradually increased, and inused in chromatographic analysis. In the chromatogram, the reaction system, when the concentrations of thethe anti-oxidation peaks were the peaks whose areas ethanol extracts of the seeds of the two buckwheatwere decreased and the anti-oxidation peaks were varieties were 30 ug mL", the dPPh free radicascanned at the full-range wavelength of 190-400 nm scavenging capacities of the ethanol extracts wereand their purities were determined according to the (51*1.5)%( Pingqiao 2)and(95*2. 1)%(Qianwei 3),retention time-recording absorption spectra.respectively. Namely, the DPPH free radical scavengingChromatographic conditions: Mobile phase A was capacities of the tartary buckwheat were significantlyethanol, and mobile phase b was water solution of higher than those of the common buckwheat at theformic acid. Gradient elution: The mobile phase changed same concentrations of the extracts(P <0.05), whichfrom A-B(35: 65)to A-B(80: 20) between 0 and 40 was related to that the total phenols content of tartarymin; The velocity of flow was 0.3 mL min, the buckwheat was higher than that of common buckwheatdetection wavelength was 360 nm and the scan was (Mo 1989; Cao et al. 2006).done at the full wavelength of 190-400 nm; and the The results of the study indicated that even ascolumn temperature was 25C.concentration was low, the extract of the tartarybuckwheat still had strong DPPH free radical scavengingDetermination of main anti-oxidative constituents effect indicating that it contained highly effective freein the ethanol extracts of buckwheat seeds by radical scavengers.HPLC-DAD-ESI-MSnHPLC identification of main anti-oxidativeMS conditions The ion source was ESI whose electro- constituents in the ethanol extracts of buckwheatspray voltage was 5 kv, the capillary temperature was seeds350oC, the velocity of N, gas was 35 pis, and the scanrange was m/z 100-800 in the negative ion modeThe ethanol extracts of the tartary buckwheatChromatographic conditions The same as those innmon buckwheat were separately mixed with DEthe section"HPLC identification of major anti-oxidationaks that the ethanol extracts of buck wheat seedssvia Pingqiao 2Data processing and statistic methods00In the study, all the data were in the form of means*standard deviation and duncan multiple comparison testwas adopted to compare the averages of the individual000factors at P<0.05RESULTSDPPH free radical scavenging effects of the中国煤化工ethanol extracts of buckwheat seedsFig. 1-cities of the ethaneextractsCN MHGentrations(differentnormal letters on the bar tops mean significant differences atFig 1 shows DPPH free radical scavenging capacities P=0.05)o2008, CAAS. A nghs meerved. Pubished by ElseMorLdAnti-Oxidative Constituents of Ethanol Extract from Buckwheat Seeds by HPLC-Electro-Spray MSsolution and the mixture was analyzed by hPlC, andthe HPLC results are shown in Figs. 2 and 3According to Fig. 2, after DPPH free radical wasadded to the ethanol extract of the common buckwheattwo chromatographic peaks decreased in area and kepttheir retention times at 3.64 and 9.50 min, and the peakcorresponding to the compound 2 disappeared, whichindicated that these compounds had reacted with DPPhfree radical so that they were main anti-oxidativeconstituents in the ethanol extract of the commonTime(min)buckwheatAfter DPPH free radical was added to the ethanolextract of the tartary buckwheat(Fig 3), the peakwhose retention time was 3. 6 min decreased in are(compound 3)and the peak with a retention time of 9. 48min nearly vanished(compound 4), which indicatedthat the ethanol extract of the tartary buckwheat con-tained at least two main anti-oxidative constituents thatwere capable of reacting with DPPH free radical andwhose anti-oxidative capacities differedDetermination of the main anti-oxidative Fig. 2 Chromatograms of the ethanol extract of the commonconstituents in the ethanol extracts of buckwheatbuckwheat before(A)and after(B)its reaction with DPPH freeradicalseeds by HPLC-DAD-ESl-MSnScanning analysis of each anti-oxidationchromatographic peak by dAD of HPLC showed thatthe spectra of compounds 1-4 were found to be verysimilar to that of quercetin, the spectra of compounds2 and 4 were found to be completely the same as that0040of quercetin, and the spectra of compounds I and 3yere completely the same as that of rutin. Theseindicated that the anti-oxidative constituents in the ethanolTime(min)extracts of buckwheat seeds might be rutin andquercetin. To further determine their structuralinformation, the study analyzed four anti-oxidativeconstituents in the ethanol extracts of buckwheat seedsby HPLC-MS and scanning them in the negative ion sTable and Figs 4 and 5 present the data, MS data,and second-order mass spectra of the anti-oxidaticonstituents in the ethanol extracts of the twobuckwheat varieties, respectively. Table shows that in中国煤化工the first-order mass spectra of compounds and 3, bothexisted in the ion form of [M-1] and could produce buck wCN MH Extract of the tartaryanions of m/z 609. To further delve into the structures radica eat bnd after(B)its reaction with DPPH free92008, CAAS. An rght reserved. Published by Elsevier UYAO Ya-pingTable Data of the four anti-oxidative constituents in the ethanol extracts of buckwheat seeds by ms in negative ion modeSampleRutin273,257,229,193.179,151,121,107273,257,229,193,179,151,121,107Qianwei 3如m609273,257,229,193.179,151,12L,107273.257,229,193,179,151.121,107s(6096),20.721.lmin#1565#15792.500且Fig 4 Second-order mass spectrum of compounds 1 and 3.MS301.7,313-318min#2352-#237631229.1250600mzFig. 5 The second-order mass spectrum of compounds 2 and 4 in negative ion modeof compounds I and 3, the study analyzed anions of comparison of the chromatographic retention times,m/z 609 by multiple-order MS. In the second-order spectral information and second-order mass spectra ofmass spectrum(Fig 4), ion of m/z 609 lost a neutral compounds 1 and 3 with those of control rutin verifiedolecule with a molecular weight of 308 and produced that compounds 1 and 3 were rutin( Yao et al. 2006).ion of m/z 301 by the action of the impacting energy, Fig. 5 presents the second-order mass spectrumhich was the same as ones formed in the first-order compounds 2 and 4. In negative ion mode, compoundsMS fragmentations of compounds 2 and 4. In the third- 2 and 4 both produced quasi-molecular ions of m/z 301order mass spectrum, ion of m/z 301 continued to and in the second-order mass spectra, ion of m/z 301fragment forming ions of m/z 273, m/z 257, m/z 229, continued to fragment producing ion fragments, whichm/z 193, m/z 179, m/z 151, m/z 121, and m/z 107, are colr山中国煤化工吗 uercetin reportewhich were the same as those in the second-order in thet al. 2007). Thechromatogram of qercetin. It follows that compounds compCNMHmass spectra ofI and 3 were derivatives of quercetin and the compounds 2 and 4 with that of control quercetin2008, CAAS. AI ights reserved Pubished by Baorun.Anti-Oxidative Constituents of Ethanol Extract from Buckwheat Seeds by HPLC- Electro-Spray Msconfirmed that the mass spectra of the two were buck wheat and common buck wheat were similarcompletely the same and thus verified compounds 2 probably belonging to similar types of compound, whichand 4 were quercetin.was related to that both of them are fagopyrum plantsElectro-spray MS analysis showed that the maDISCUSSIONnti-oxidative constituents in the ethanol extracts ofbuckwheat seeds were rutin and quercetin, whichBecause people are afraid that man-made anti-oxidants agreed with what had been reported (Yen et al. 1996;have side-effects. more and more attention has been Guo et al. 2004; Zhang et al. 2003). Although thepaid to identifying high-activity anti-oxidants from buckwheat contains a great amount of flavonoid,rutinnatural foods. Currently, the most common methods and quercetin are the major ones amongst them. Andfor identifying anti-oxidative constituents from natural the anti-oxidative activity of quercetin is higher thanextracts is to extract, separate, and purify anti-oxidantsthat of rutin, for the rutinose takes the place of hydroxylin the natural extracts and evaluate their relevant group at C] of carboatomic ring of rutin, and the hydroxylbioactivities. but the method is time- and labor. group at C, of the carboatomic ring plays an importantconsuming and liable to cause antioxidative constituents role in enhancing the anti-oxidative activities ofto disintegrate during its operation, thus possibly flavonoids(Liu and Zhu 2007: Heim et aL. 2002, Ericaffecting its reliability.et al. 1999). The study only investigates the main antip according to Fig. 1, the ethanol extracts of buckwheat oxidative constituents in the ethanol extracts ofseeds were able to effectively scavenge dpph fro buckwheat seeds, but because the anti-oxidativeradicals and their DPPH free radical scavenging constituents of buck wheat seeds differ in polarity andcapacities appeared dosage dependent. At the samtheir solubilities differ amongst different solvents, it isconcentrations, the ethanol extract of the tartary necessary to do an in-depth study cof the active anti-buckwheat seeds had remarkably higher DPPH free oxidative constituents in buckwheat seeds by modemradical scavenging capacities than that of the common analytic methods so as to provide a basis for thebuckwheat seeds, which was possible to relate to the exploitation and utilization of buckwheat resourcesconcentrations of the anti-oxidative constituents in theethanol extract of the tartary buckwheat seeds(the data CONCLUSIONare not presented). But because the ethanol extract ofbuckwheat seeds has a complex composition and theBy HPLC-ESI-MS the study set up a new method forkinds and contents of its anti-oxidative constituents are identifying anti-oxidative constituents in the ethanolunknown, DPPH free radical-based evaluation is ableextract of buckwheat seeds. The results indicated thatto reveal the activity of anti-oxidative constituents rutin and quercetin were the main anti-oxidativeotained by mixed extraction but unablole to reveal the constituents in the ethanol extracts of both tartarycontents and relative anti-oxidative capacities of mixedbuckwheat and common buckwheat and this methodanti-oxidative constituents. HPLC-DAD analysis was characterized by fastness, simplicity, convenience,showed that after dPPh free radical was added to theand high flux in researching on anti-oxidative constituentsethanol extract of buckwheat seeds. some chromatographic peaks lowered their areas and evendisappeared. This phenomenon indicated that theReferencescompounds that the peaks corresponded to had reactedwith DPPH free radical, producing new compoundsCao w, Chen wJ, Yao Y P. 2006. 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