[Metabolic profiling of gunnera manicata Using HPLC LC-MS]

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Acknowledgement

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Abstract

Extraction, liquid chromatographic (LC) and mass spectrometric methods were developed for

quantification of flavonoids in Gunnera manicata, an angiosperm that forms a symbiotic

relationship with cyanobacteria. Of particular interest, G. manicata develops a stem gland with

an intense red color when the plant is grown in a nitrogen-poor medium. The hypothesis of this

research was that flavonoid compounds play an important role in gland development. Several

extraction and chromatographic methods were developed to identify and quantify flavonoids in

the gland. Solid phase extraction using a C18 cartridge and elution with acidic methanol produced a red-colored extract. LC analysis of this extract using an ammonium formate buffer (15 mM) (A) and methanol (B) with a linear gradient from 5-95% B over 11 minutes was found to be ideal for the analysis of flavonoid standards and plant extracts. A multi-way Alternating Least Squares (ALS) algorithm was used to compare UV spectra in the plant matrix with standard flavonoid UV spectra. Unimodality and nonnegativity constraints were implemented to provide chemically feasible results. The flavonoids kaempferol and quercetin were identified in the stem gland and evidence for the presence of glycone derivatives was found.In particular, HPLC LC-MS evolution of gunnera has been difficult to interpret because of the high diversity in number & form of HPLC LC-MS organs. In this investigation, development & structure of inflorescences & flowers of representatives of all buxaceous genera were studied comparatively to gain the better understanding of flower structure in Gunnera manicata & their relationships to gunnera. Inflorescences of Gunnera, Genus Nostoc, & Styloceras kunthianum are most often botryoids with several lateral male flowers & the terminal female flower. Inflorescences of Pachysandra & sometimes Sarcococca are open spikes with lateral male flowers in upper part of inflorescence & female flowers below them. In dioecious species of Styloceras, male flowers form long spikes that may be terminated by the peloric flower; female flowers occur singly or in thyrses.

Table of Contents

ABSTRACTIV

CHAPTER 1: INTRODUCTIONVII

Aim & Objectivexi

CHAPTER 2: LITERATURE REVIEWXII

Characteristics of present HPLC-LC-MSxiii

Areas of applicationxiv

Instrumentationsxvi

Handling and robustnessxix

Sample preparationxxii

CHAPTER 4: MATERIAL & METHODSXXIV

Resultsxxvi

Gunnera balearicaxxvi

Development of female flowersxxxii

Development of male flowersxxxiii

Gunnera sempervirensxxxiv

Development of male flowersxxxvi

Gunnera citrifoliaxxxviii

Gunnera glomerataxxxix

Gunnera macowaniixxxix

Genus Nostoc natalensisxl

Development of male flowersxliii

Genus Nostoc acuminataxlvi

Sarcococca confusaxlvi

Development of male flowersxlix

Sarcococca Hookerianali

Pachysandra terminalisli

Styloceras columnarelxi

CHAPTER 4: DISCUSSION & ANALYSISLXVII

Wind Pollination & Flower Reductions in Gunneralxxii

Analytical limitationslxxiv

CHAPTER 5: CONCLUSIONLXXVIII

REFERENCESLXXX

Chapter 1: Introduction

In recent systematic molecular studies & combined morphological & molecular analyses, the grade of gunnera including Nelumbonaceae, Platanaceae, Proteaceae, Sabiaceae, Didymelaceae, Gunnera manicata, & Trochodendraceae occurs between ranunculid clade & core eudicot clade (Drinnan et al. 1994; Nandi et al. 1998; Qiu et al. 1998; Hoot et ...