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Separation and Quantitation
Of Kava Lactone Yielding Precursor(s) From
Piper methysticum Forst.
A Thesis Submitted in Fulfillment of the Requirements for theDegree of
Master of Science
Sunny Yurendra Prasad(BSc- Biology and Chemistry, USP)
Division of Chemistry
School of Biological, Chemical and Environmental Sciences
Faculty of Science and Technology
UNIVERSITY OF THE SOUTH PACIFIC
Suva, Fiji Islands
Part of the work described in this thesis has been presented and submitted as:
1. Naiker, M., Prasad, S. Y. (2005). Detection of kava lactone-yielding precursor(s)
in kava (Piper methysticum Forst.) roots. In: The 12th Royal Australian Chemical
Institute (RACI) Convention, Sydney Convention and Exhibition Centre, Darling
Harbour, Australia, 3-7th July, 2005. Abstract book pp 292.
2. Naiker, M., Prasad, S. Y., Singh, R. D., Singh, J. A., Voro, T. N (2006).
Investigation of kava lactone-yielding precursor(s) occurrence in kava roots.
Submitted in The Natural Product Communication.
Table of Contents
Declaration iiAcknowledgements iii
List of Figures vList of Tables vi
List of Abbreviations vii
1. General Introduction 1
2. Literature Review 5
2.1 The Kava Plant 5
2.2 The Bioactive Principle and its Effects 6
2.3 Other Isolated Compounds from Piper methysticum Forst. 92.3.1 Alkaloids 92.3.2 Glutathione 102.4 Occurrence of Polar Precursors in Plant Tissues 112.4.1 Formation and Role of Glycosides 122.4.2 Composition of Glycosidic Precursors in Plant Tissues 172.4.3 Analysis of Intact Glycosidic Precursors 192.4.4 Analysis of Aglycones after Hydrolysis of Glycosides 212.5 Analytical Techniques Involved in Detection and Characterization ofKava
2.5.1 HPLC Analysis 242.5.2 UV Analysis 252.5.3 NMR Analysis 26
3. Isolation and Purity Determination of the Major Kava
3.1 Introduction 27
3.2 Results and Discussion 273.2.1 Extraction and Removal of Free Kava Lactones 273.2.2 Detection and Isolation of Pure Kava Lactones 303.2.3 Purity Determination of Isolated Kava Lactones 31
3.2.3a HPLC Analysis 313.2.3b UV Analysis 323.2.3c 1HNMR Analysis 34
3.3 Conclusion 39
4. Refluxing at Various Aqueous pHs and EnzymaticHydrolysis of the Lactone Depleted (delactoned) Residue
4.1 Introduction 404.2 Results and Discussion 41
4.3 Conclusion 47
5. Fractionation and Aqueous pH Reflux of the LactoneDepleted Residue
5.1 Introduction 48
5.2 Results and Discussion 495.2.1 Thin Layer Chromatographic Analysis of the Delactoned Residue 495.2.2 Reverse Phase Analytical HPLC Analysis of the Delactoned Residue 495.2.3 Reverse Phase Fractionation and Aqueous pH Reflux of the Delactoned 50
5.2.4 Precursor Essay 525.2.5 1HNMR Analysis of Precursor Rich Fractions 555.3 Conclusion 57
6 Methodology 596.1 General 59
6.2 Isolation and Purity Determination of the Major Kava Lactones 616.2.1 Sample 616.2.2 Aqueous Extraction 616.2.3 Removal of Free Kava Lactones 616.2.4 Detection and Isolation of Pure Kava Lactones 626.2.5 Purity Determination of the Isolated Kava Lactones 626.3 Refluxing at Various Aqueous pHs and Enzymatic Hydrolysis of the
6.3.1 Reflux of the Delactoned Residue at Various Aqueous pHs 636.3.2 Enzymatic Hydrolysis of the Delactoned Residue 646.3.3 Extraction of Regenerated Kava Lactones 64
6.3.4 HPLC Analysis of Regenerated Kava Lactones 64
6.4 Fractionation and Aqueous pH Reflux of the Delactoned Residue 656.4.1 Development of Analytical Parameters for Reverse Phase HPLC Analysis
184.108.40.206 Thin Layer Chromatographic Analysis 6220.127.116.11 Reverse Phase Analytical HPLC Analysis 656.4.2 Reverse Phase Semi-preparative HPLC Analysis and Fractionation 666.4.3 Aqueous pH Reflux of Individual Reverse Phase Fractions 676.4.4 1HNMR Analysis of Individual Reverse Phase Fractions 67
7 References 68
8 Appendix 75
Kava lactones are the biologically active ingredients in Piper methysticum Forst. (kava
plant). Apart from their existence in free forms, it was found that a portion of these
biologically active kava lactones exist in bound forms. Work presented in this thesis
reports for the first time the existence of kava lactone-yielding precursors for
desmethoxyyangonin, yangonin and kavain in dried kava roots.
After the removal of all free desmethoxyyangonin, yangonin and kavain from the polar
root extracts, a second crop of desmethoxyyangonin, yangonin and kavain was observed
when subjected to various aqueous-pH reflux and enzymatic hydrolyses. This result
suggests the occurrence of kava lactone-yielding precursor(s).
Further purification of the delactoned residue using semi-preparative HPLC resulted in
separation of six chromatographically distinguishable precursor-rich fractions. Each
fraction when boiled in pH 5-buffered water yielded different ratios of
desmethoxyyangonin, yangonin and kavain. This suggests the possible existence of
multiple precursors for each kava lactone, which are structurally/chemically different.
Furthermore, 1HNMR spectroscopy of the fractions provided evidence for glycosides.
Due to the initial extraction of the kava sample with water, it is believed that the kava
extract could to be rich in glycosides and from the results obtained using 1HNMR
spectroscopy it was evident that the precursors of kava lactones are glycosides, however
further purification was necessary for structural elucidation which was beyond the scope
of this research.
I, Sunny Yurendra Prasad, hereby testify that the material contained in this thesis has not
been published elsewhere, except where due reference is made, and that the thesis has not
been used for the award of any degree or diploma of a university or other institute of
higher learning in any institution.
Sunny Yurendra Prasad
Dr Mani Naiker
Dr Tevita N. Voro
Firstly I would like to take this opportunity to thank my supervisors Dr Mani Naiker and
Dr Tevita N. Voro (Division of Chemistry, School of Biological, Chemical and
Environmental Sciences, University of the South Pacific) for their advice, encouragement
and criticism. I am thankful to them for having faith in me during the course of this study.
Special thanks to Dr David Tucker of University of New England, Armidale, NSW,
Australia for his valuable time in performing NMR analysis.
Miss Ranjeeta Devi Singh (Post graduate student, Faculty of Science and Technology,
USP) is thanked for her help and being such a nice colleague.
Mr Edward Narayan of USP is duly acknowledged for his assistance with formatting and
going over the final draft of this thesis.
I would like to thank Mr Lawrence Narayan and Mr Lasarusa Donu of USP for their help
in organizing the chromatograms.
I would like to thank Mr Steve Sutcliffe (Chief technician, Division of Chemistry, USP),
Mr Vas Deo (Senior Technician, Division of Chemistry, USP) and all the technical staff
of the Division of Chemistry, USP, for their support and time over the duration of this
My colleagues at the Faculty of Science and Technology (USP) are thanked in particular
Mr Mohammed Shereez Ali, Mr Anand Chandra, Mr Shaneel Chandra, Mr Sachin Singh,
Mr Amit Sukaal, Mr Vimlesh Chand, Mr Alovaka Fuli, Miss Joshlin Singh, Miss Kavita
Ragni, Miss Vikashni Nand, Miss Riteshma Singh, Miss Kirti Patel, Miss Ranjani Devi,
Miss Radhika Singh, Miss Heena Lal and Miss Irene Hanson.
The NZAID and SPAS Research Committee is acknowledged for their funding during the
time of my study.
Furthermore, I would like to take this opportunity to thank my friends in particular Mr
Salen Rao, Mr Elvin Rao, Mr Ravinesh Chand, Miss Kirti Mala and Mr Salvindra Pillay
for their encouragement, invaluable support and making my study enjoyable.
Mr & Mrs Rajan Murti and Mr & Mrs Salendra Prasad are duly thanked for their support
during the latter years of this study.
I would like to thank Mrs Pawan Naiker for her support and guidance throughout this
Special thanks to my partner Malini and best mate Dinesh D Gosai for their
understanding and companionship during the course of this work.
Finally, I would like to give my sincere gratitude to my parents (Mr & Mrs Birendra
Prasad), my brother (Nicky) my sister (Anjanita), my brother-in-law (Bijesh) and the rest
of my family and friends for their patience, encouragement and their contribution to my
List of Figures
Figure 1.1. Keto-enol tautomerism of kavain prior to glycosylation
Figure 2.1. Structures of the six major kava lactones
Figure 2.2. General transglycosylation scheme 13
Figure 2.3. Glycosylation pathway for geraniol 14
Figure 2.4. Proposed pathway for the glycosylation/hydrolysis of kavain 15
Figure 2.5. Proposed pathway for the glycosylation/hydrolysis
Figure 3.1. HPLC chromatograms obtained for crude kava lactone extract and
kava lactone standards
Figure 3.2. U