GLYCOSIDES IN Viola Tricolor L.

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CHAPTER 3: MATERIAL AND METHODS follows: source voltage 1.7kV, capillary temperature 220 ◦ C, capillary voltage 41V, tube lens 115V; in the negative mode: source voltage 1.3kV, capillary temperature 220 ◦ C, capillary voltage -50V, tube lens -152V . Data acquisition and interpretation wasdone with the Xcalibur software from ThermoFisher. 3.9 NMR spectroscopy 18mg Fraction E was dissolved in 1ml DMSO-d6 and characterized on a two- channel Varian Inova 600MHz (Palo Alto, CA) NMR spectrometer equipped with a waveform generator, a pulsed field gradient (PFG) unit and a dual in- verse broad-band probe-head. In addition to registering the 1 H-NMR and 13 C- NMRspectrastandard2Dexperiments(COSY,TOCSY,NOESY,HSQC,HMBC) and selective 1D TOCSY-TOCSY and TOCSY-NOESY experiments were ap- pliedforassignment.AllspectrawerecalibratedtointernalTMS(tetra-methyl- silane). 3.10 Anthocyanidinandflavonoidcontent Theantocyanidin andflavonoid content ofthe driedsampleswere determined by applying the methods of the European Pharmacopoeia 5.0, paragraph ’Bil- berryfruit,fresh’(Myrtillifructusrecens)and’Wildpansy’(<strong>Viola</strong>etricolorisherba) [143]. 3.11 Invitroantioxidantassays TEACassay ABTS(2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid)was dissolvedinwaterin7mMconcentration. Itsradicalmonocationwasproduced by reacting the ABTS solution with 2.45mM (final concentration) potassium persulfate and letting the mixture stand in dark at room temperature for at least12hbeforeuse. TheABTS •+ stocksolutionwasdilutedwithspectroscopic grade ethanol to 0.9 absorbance unit at 734nm. At least four different volumes of the diluted sample were added to 2.5ml ABTS solution resulting in different final concentrations and producing inhibition of the blank solvent between 20% - 80%. Absorbance values were measured at 734nm after 0, 0.5; 0.66; 0.83; 43
CHAPTER 3: MATERIAL AND METHODS 1; 1.5; 2; 2.5; 3; 4; 5 and 6 minutes. For the determination of the so-called inhibition percentage, weextrapolated thefinal absorbancebynumericallysolving the simplest possible reaction kinetics model. The inhibition percentage pro- duced by a given sample concentration was calculated as (100-At/A0)*100 (At: theextrapolatedfinalabsorbance,A0: theabsorbanceoftheblanksolvent). The antioxidant activity was characterized by plotting the inhibition percentage of thesamplesasafunctionofsampleconcentrationfollowedbylinearregression (data not shown). The concentration resulting in 50% inhibition is referred to asIC50 value. DPPH assay DPPH • was dissolved in HPLC grade methanol in 0.25g/L concentration. The DPPH • stock solution was diluted with HPLC grade methanol to 0.9 absorbance unit at 515nm. At least four different volumes of the diluted sample were added to 2.5ml DPPH • solution resulting in different final concentrations and producing inhibition of the blank solvent between 20% - 80%. Absorbancevaluesweremeasuredat515nmafter0,0.5,0.66,0.83,1,1.5,2,2.5, 3, 4, 5, 6 minutes. The inhibition percentage was calculated as for the TEAC assay. 3.12 Fortifiedsamplerecoverytest 1.2mL of diluted stock solution (prepared according to Method 3) was further diluted with 0.8mL 70 % methanol. This aliquot served as a blank solution in the fortified sample recovery test. Another 1.2mL aliquot of the stock solution was diluted with 0.8mL rutin solution (267 µg/mL, 70% MeOH), corre- sponding to the addition of 213.6 µg rutin. The recovery (R) was calculated as R = 100(mfound-minitial)/madded; (mfound: rutin content in the fortified sample; minitial: rutin content in the blanksample;madded: addedrutin amount). 44
Page 1 and 2: ANTIOXIDANT FLAVONOID GLYCOSIDES IN
Page 3 and 4: Összefoglaló Avadárvácska(Viola
Page 5 and 6: CONTENTS 3.10 Anthocyanidin and fla
Page 7 and 8: CONTENTS R Recoveryin the fortified
Page 9 and 10: CHAPTER 1: PRELIMINARIES AND AIMS O
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Page 13 and 14: CHAPTER 2: INTRODUCTION Table2.1: S
Page 15 and 16: CHAPTER 2: INTRODUCTION A number of
Page 17 and 18: CHAPTER 2: INTRODUCTION charides, v
Page 19 and 20: 14 Figure 2.4: Biosynthesis ofthe m
Page 21 and 22: CHAPTER 2: INTRODUCTION or acetonit
Page 23 and 24: CHAPTER 2: INTRODUCTION 2.4.1 Instr
Page 25 and 26: CHAPTER 2: INTRODUCTION Figure 2.6:
Page 27 and 28: CHAPTER 2: INTRODUCTION cyanidins w
Page 29 and 30: CHAPTER 2: INTRODUCTION Table 2.2:
Page 31 and 32: CHAPTER 2: INTRODUCTION Figure 2.8:
Page 33 and 34: CHAPTER 2: INTRODUCTION icant Y −
Page 35 and 36: CHAPTER 2: INTRODUCTION Figure 2.10
Page 37 and 38: CHAPTER 2: INTRODUCTION ide) more e
Page 39 and 40: CHAPTER 2: INTRODUCTION dance for b
Page 41 and 42: CHAPTER 2: INTRODUCTION hand, contr
Page 43 and 44: CHAPTER 3 Material and methods 3.1
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Page 71 and 72: 66 Table 4.3: Principal ESI-MS/MS p
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Page 81 and 82: 76 Table 4.6: The antocyanidin and
Page 83 and 84: CHAPTER 5 Conclusion Although heart
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Page 87 and 88: CHAPTER 5: CONCLUSION UIBK-HorvathL
Page 89 and 90: BIBLIOGRAPHY insulinresistanceanddi
Page 91 and 92: BIBLIOGRAPHY [28] N. Saito, C. F. T
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Listof publications Papersrelatedto
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BIBLIOGRAPHY The antioxidant activi
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was evaporated to dryness under red
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151, 135, 119, and 103 ( 1,3 B + )
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Anal Bioanal Chem DOI 10.1007/s0021
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Anal Bioanal Chem The eluate was co
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Anal Bioanal Chem Column chromatogr
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Anal Bioanal Chem a HO OH d HO OH O
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Anal Bioanal Chem 3. Witkowska-Bana
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12 V. Vukics et al. / J. Chromatogr
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Table 2 Principal ESI-MS/MS product
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