GLYCOSIDES IN Viola Tricolor L.

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CHAPTER 4 Resultsand discussion 4.1.1 Basicextractionmethod 4.1 Scoutinganalyses As the traditional internal administration of heartsease herb is as a tea, i.e., by preparingan infusion, in thisthesis weprimarily aimedatthe thorough analyses of this aqueous extract. Aqueous solutions, however, are microbiologically instable and difficult to handle (e.g. to evaporate). Theoretically, the appli- cation of methanolic solutions could alleviate these drawbacks. The samness of the heartsease methanolic and aqueous extracts’ compositions was demonstrated by HPLC analyses (data not included). In addition, the effectiveness of methanol as extraction solvent was also verified by TLC expreiments. Sam- plesobtained by consecutive extraction with n-hexane, dichloromethane, ethyl acetate, and 70% methanol (Method 1) were compared. The compositions of extractswerecharacterizedbythinlayerchromatography. TLCplateswerede- veloped in systems of different polarities (TLC Methods 1-3) suitable for the analysis of highly apolar compounds as well as flavonoid aglycones and glycosides, and attended with special color reagents such as Naturstoff. Results clearly demonstrated that the apolar components of heartsease were extracted by the most apolar solvent, n-hexane. In addition, the methanolic extract was enriched in components of similar polarity of flavonoid glycosides and aglycones, which are supposed to be present also in the aqueous extract. Hence- forth, in most sample preparation methods after a pre-extraction with chlo- roform, producing Fraction A, heartsease herb was extracted with methanol, yieldingFraction B(Method 2)(Fig. 4.1). 45
CHAPTER 4: RESULTS AND DISCUSSION 4.1.2 Plantmaterials Asapart of our preliminary examinations, plant materialsfrom three different sources were compared (Sec. 3). The first sample was collected and identified in Transilvania by Prof. Kalman Csedo (Babes-Bolyai University). The second and third samples were purchased from Fitopharma Ltd. and MDR 2000 Ltd., respectively. Our HPLC fingerprint analyses revealed that the compositions of thecollectedandpurchasedsamplesexhibitedonlyslightdifferences. Thus,for further –especially quantitative– analyses the sample easisiest to obtain (from Fitopharma Ltd.) was used. The similarity of the compositions of collected andpurchasedplantsampleswassupportedbylaterMSexperiments(datanot included). 4.2 Preparativeseparationof FractionB For the preparative separation of crude plant extracts, conventional column chromatography methods are widely used. To partition polar components, such as flavonoids or other phenoloids, stationary phases of polyamide, cel- lulose, silica gel and Sephadex LH-20 were mostly reported [144], most often using aqueous alcoholic solutions (methanol-water or ethanol-water) for their elution. Due to the good light absorption of flavonoids and the fluorescence propertiesoftheirderivatives[85],theirchromatographicseparationcaneasily be detected. The exact composition of the fractions are usually determined by analytical tools of HPLC, LC-MS, LC-NMR, CE and CEC [82, 145]. However, thesemethodsoften requiretheaidofreferencestandard molecules. Ifneeded, a second separation step of column chromatography, preparative TLC or even preparative HPLC can be utilized. The preparative separation of the methanol extract of heartsease (Fraction B) was carried out first by Sephadex LH-20 column chromatography. In the preliminary experiments, fractions of identical volumes were collected and the composition of the fractions was screened by TLC (TLC Method 1). Based on the TLC results a fractionation protocol was developed described in Material and methods (Sec. 3) anddelineated in Fig. 4.1. This fractionation method pro- vided Fractions C-G, which together with Fraction B were analyzed by HPLC (Fig. 4.2a-f). The chromatograms were achieved as a result of gradient elution from a RP-C18 column detected at UV 340 nm, a distinctive wavelength for 46
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Page 3 and 4: Összefoglaló Avadárvácska(Viola
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Page 7 and 8: CONTENTS R Recoveryin the fortified
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Page 13 and 14: CHAPTER 2: INTRODUCTION Table2.1: S
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Page 31 and 32: CHAPTER 2: INTRODUCTION Figure 2.8:
Page 33 and 34: CHAPTER 2: INTRODUCTION icant Y −
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Page 41 and 42: CHAPTER 2: INTRODUCTION hand, contr
Page 43 and 44: CHAPTER 3 Material and methods 3.1
Page 45 and 46: CHAPTER 3: MATERIAL AND METHODS 3.5
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Page 83 and 84: CHAPTER 5 Conclusion Although heart
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Page 89 and 90: BIBLIOGRAPHY insulinresistanceanddi
Page 91 and 92: BIBLIOGRAPHY [28] N. Saito, C. F. T
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BIBLIOGRAPHY electrospray ionizatio
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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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