Anthocyanins in selected Proteaceae

Schmeisser, Michael (2002-12)

Thesis (MScAgric)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: Trials were conducted to follow colour development during the growth season of the commercial Leucadendron cultivar 'Siivan' Red', as well as the Protea cultivars 'Ivy', 'Carnival' and 'Sylvia'. HPLC analysis showed that pigments responsible for red colour in Leucadendron and Protea cultivars are anthocyanins. Initial extraction of anthocyan ins from freeze-dried and finely ground Leucadendron leaves, using 5% formic acid in methanol, resulted in the precipitation of gel-like compounds, making the extracts impossible to filter for HPLC. Trials were conducted to determine the optimum extraction solvent and extraction time for anthocyanin quantification in Leucadendron, using 'Safari Sunset' (Leucadendron sa/ignurn x L. /aureo/urn) as reference material. Acetone, 80% acetone, 5% formic acid in water and 5% formic acid in methanol were added to freeze dried leaf samples and allowed to extract for one hour. Extracts were analysed by reverse-phase HPLC. Extractions with methanol and water yielded extracts with a high content of pectin-like compounds, making them difficult to purify for HPLC. 80% acetone proved to be the most efficient extraction solvent, yielding the highest anthocyanin concentration, and showing the least amount of insoluble compounds. Optimal extraction time was determined for 80% acetone by testing 1, 6 and 24-hour extractions respectively. Although 6 hour extraction showed a slightly higher yield, a 1-hour extraction should suffice for quantification of anthocyanin changes over time, as trends will clearly be evident. 'Siivan Red' shoots meeting export quality can generally be harvested from mid February to mid April (late summer to autumn). The harvesting period ends as the colour of the distal leaves forming the flower head change from red to green. Anthocyanin, chlorophyll and carotenoid fluctuations were investigated in 'Siivan Red' during the 2000 growth season. The observed red colour loss is the result of the net degradation of anthocyanins and appeared to be correlated to the phenological development of the shoot. Although a positive correlation was noted between anthocyanin content and average daily temperature and mean daily sunlight hours, it appears unlikely that they are the causal factors for the observed colour change. Anthocyanin degradation started during conditions of long exposure to high light intensities and continued during a period of low temperature (autumn), both which are known to favour anthocyanin synthesis rather than its degradation. Therefore it appears that colour development in 'Silvan Red' is developmentally regulated. The Leucadendron cultivar 'Safari Sunset' with the same parentage as 'Siivan Red', is noted for its more intense red-purple colouration, which it does not lose during its development. The more intense colouration of 'Safari Sunset' has been ascribed to the higher total anthocyanin concentration, which was almost double that encountered in 'Siivan Red'. 'Safari Sunset' was shown to contain the same major types of anthocyanins, and hence the purple colouration (not seen in 'Siivan Red') can not be explained in terms of different types of anthocyanins being present. However, the ratio between the two major anthocyanins present in both 'Safari Sunset' and 'Siivan Red' were significantly different in that 'Safari Sunset' showed a considerably higher concentration of peak 1, which is most likely responsible for the observed purple colouration. High anthocyanin concentrations have been noted to buffer against visual changes in colour, which is the most probable reason that a colour loss is not observed in 'Safari Sunset'. Colour development of the innermost involucral bracts of three Protea cultivars was followed from an initially selected inflorescence size to commercial harvest (when flowers have opened slightly). 'Ivy', 'Carnival' and 'Sylvia' show a light pink, dark pink and red colouration respectively, which has been ascribed to differences in total anthocyanin concentration. Colour development in 'Carnival' showed a quadratic trend with time, with the highest rate of anthocyanin synthesis occurring a week prior to harvest. Colour development in 'Ivy' and 'Sylvia' were shown to be linear with time. As inflorescences are closed during their development and anthocyanin synthesis in the innermost involucral bracts occurred in darkness, light does not appear to have a major influence on colour development. Temperature did not appear to have a significant effect on flower colour, as flowers developing later in the season, when ambient temperatures were lower, showed no significant differences in anthocyanin concentration to those harvested earlier. Hence, it appears that colour development in all three cultivars is developmentally regulated.

AFRIKAANSE OPSOMMING: Hierdie studie is onderneem om kleurontwikkeling van die Leucadendron kultivar 'Silvan Red' en die Protea kultivars 'Ivy', 'Carnival' en 'Sylvia' te ondersoek. Die rooi kleur van distale 'Siivan Red' blare en Protea omwindselblare is te wyte aan antosianiene. Eerstens is gepoog om die ektraksie van antosianiene vir kwantifisering deur hoë druk vloeistof chromatografie (HPLC) te verfyn. Aanvanklik is 5% mieresuur in metanol gebruik om antosianien vanuit gefriesdroogde en fyngemaalde Leucadendron blare te ekstraheer. 'n Gelagtige neerslag het dit egter onmoontlik gemaak om die ekstrak te filtreer. Vervolgens is eksperimente uitgevoer om die geskiktheid van asetoon, 80% asetoon, 5% mieresuur in water en 5% mieresuur in metanol as alternatiewe vir 5% mieresuur in metanol te bepaal. Antosianiene is deur middel van HPLC gekwantifiseer na 'n uurlange ekstraksie vanuit 'Safari Sunset' (Leucadendron sa/ignurn x L. /aureo/urn) blaarmonsters. 'n Onoplosbare neerslag van onsuiwerhede na ekstraksie met metanol en water het filtrasie bemoeilik. 80% asetoon was die doeltreffendste ekstraheermiddel deurdat dit beide die hoogste opbrengs van antosianiene en die minste onsuiwerhede gelewer het. Vervolgens is die optimale duur van antosianienekstraksie met 80% asetoon bepaal deur vir een, ses en 24 uur te ekstraheer. Resultate het getoon dat, alhoewel ses uur ekstraksie 'n effense hoër antosianienkonsentrasie lewer, 'n uur voldoende behoort te wees vir kwantifisering van antosianien. Uitvoergehalte 'Siivan Red' lote word gewoonlik vanaf middel Februarie tot middel April (Iaat somer tot herfs) geoes. Die oestyd eindig met 'n verandering in die kleur van distale blare van rooi na groen wat die bemarkbaarheid van lote verlaag. Ten einde hierdie kleurverandering te kwantifiseer is veranderinge in die konsentrasies van antosianien, chlorofiel en karotenoide in distale 'Siivan Red' blare gedurende die 2000 groeiseisoen gemeet en in verband gebring met omgewingstoestande en ontwikkelingstadiums. Rooi kleurverlies van distale 'Silvan Red' blare hou klaarblyklik verband met 'n geleidelike afname in hul antosianienkonsentrasies vanweë netto degradasie. Veranderinge in die kleur en antosianienkonsentrasie van blare het saamgeval met spesifieke ontwikkelingsperiodes. Kleurveranderinge in 'Siivan Red' kon nie met omgewingstoestande verbind word nie. Kleuronwikkeling word klaarblyklik intern gereguleer om saam te val met spesifieke ontwikkelingstadia. Die Leucadendron kultivars 'Safari Sunset' en 'Siivan Red' het dieselfde ouers. Eersgenoemde kultivar het egter In intenser rooi-pers kleur wat ook stabiel blyoor die hele groeiseisoen. Die intenser kleur kan toegeskryf word aan die bykans twee keer hoër antosianienkonsentrasies wat 'Safari Sunset' blare oor die groeisoen handhaaf. Hierdie hoër antosianienkonsentrasie is moontlik ook die rede vir die oëskynlik groter kleurstabiliteit van 'Safari Sunset' aangesien hoë pigmentvlakke kleur buffer teen veranderinge in pigmentkonsentrasie. 'Safari Sunset' se pers skynsel is moontlik te wyte aan die relatief groter bydrae van piek 1 op die chromatogram tot die totale antosianienkonsentrasie. Andersins het die twee kultivars 'n soortgelyke antosianienprofiel. Ten einde die regulering van kleurontwikkeling in Protea te ondersoek, is antosianienkonsentrasies in die binneste bloeiwyse-omwindelsblare van die Protea kultivars 'Ivy', 'Carnival' en 'Sylvia' gemeet vanaf 'n pre-geselekteerde blomgrootte tot en met kommersiële oestyd wanneer die blomme begin oopgaan. Die drie kultivars wisselonderskeidelik in kleur van lig pienk en donker pienk tot rooi. Hierdie kleurverskille was te wyte aan verskille in antosianienkonsentrasie. Antosianienkonsentrasies in 'Ivy' en 'Sylvia' het lineêr toegeneem oor tyd, terwyl in die geval van 'Carnival', maksimum antosianienvlakke reeds 'n week voor oes bereik is. Blomme wat by laer temperature later in die seisoen of vroeër by hoër temperature ontwikkel het, het nie betekenisvol verskil in antosianienkonsentrasie nie. Gevolglik is lig en temperatuur klaarblyklik van mindere belang vir Protea kleurontwikkeling. Soos met Leucadendron die geval was, word kleurontwikkeling in Protea dus ook intern gereguleer.

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