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

By

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

February, 2006

Publications

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

PageAbstract i

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

Lactones

24

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

Lactones

27

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 pH’s and EnzymaticHydrolysis of the Lactone Depleted (delactoned) Residue

40

4.1 Introduction 404.2 Results and Discussion 41

4.3 Conclusion 47

5. Fractionation and Aqueous pH Reflux of the LactoneDepleted Residue

48

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

Residue

5.2.4 Precursor Essay 525.2.5 1HNMR Analysis of Precursor Rich Fractions 555.3 Conclusion 57

5.4 Outlook

�

58

6 Methodology 59

6.1 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 pH’s and Enzymatic Hydrolysis of the

Delactoned Residue

63

6.3.1 Reflux of the Delactoned Residue at Various Aqueous pH’s 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

Delactoned Residue

65

6.4.1.1 Thin Layer Chromatographic Analysis 656.4.1.2 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

i

Abstract

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.

ii

Declaration

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

. ………………………

Supervisor

Dr Mani Naiker

……………………...

Supervisor

Dr Tevita N. Voro

iii

Acknowledgements

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

study.

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.

iv

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

project.

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

education.

v

List of Figures

Page

Figure 1.1. Keto-enol tautomerism of kavain prior to glycosylation

Figure 2.1. Structures of the six major kava lactones

3

7

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

of desmethoxyyangonin

15

Figure 3.1. HPLC chromatograms obtained for crude kava lactone extract and

kava lactone standards

29

Figure 3.2. UV spectra of DMY, YAN and KAV 32

Figure 4.1. HPLC chromatograms obtained for various aqueous pH reflux and

enzyme hydrolysate

42

Figure 4.2. % of DMY, YAN and KAV regenerated under various reflux

conditions and enzymatic hydrolysis of the totally delactoned residue

45

Figure 5.1. HPLC chromatogram for the delactoned residue 50

Figure 5.2. HPLC chromatograms obtained for pH 5 reflux of precursor rich

Fractions

51

Figure 5.3. % of DMY, YAN and KAV regenerated at pH 5 reflux for the

precursor fractions

53

vi

List of Tables

Page

Table 3a. Retention time for the three kava lactones 30

Table 3b. Comparison of retention times for the isolated kava lactones 31

Table 3c. Physical constants for kava lactone standards 33

Table 3d. 1HNMR data for DMY 35

Table 3e. 1HNMR data for YAN 36

Table 3f. 1HNMR data for KAV 37

Table 4a. Peak area and DRF for DMY, YAN and KAV standards 43

Table 5a. The ratios of DMY, YAN and KAV regenerated (μg) per kg of

powdered kava

54

vii

List of Abbreviations

CCC Countercurrent chromatography

DCCC Droplet countercurrent chromatography

DCM Dichloromethane

DHK Dihydrokavain

DHM Dihydromethysticin

DMY Desmethoxyyangonin

DNA Deoxyribonucleic acid

DRF Detector response factor

GC Gas chromatography

GC-MS Gas chromatography - mass spectroscopy

G-G Glycosyl-glucose

G-LC Gas liquid chromatography1HNMR Proton nuclear magnetic resonance

HPLC High performance liquid chromatography

IR Infrared spectroscopy

KAV Kavain

KHP Potassium hydrogen phthalate

LC-MS Liquid chromatography - mass spectroscopy

LG Liquid gas

METH Methysticin

MLCCC Multi layer countercurrent chromatography

MS Mass spectroscopy

NDP-Carbohydrate Nucleotide-activated carbohydrate

NMR Nuclear magnetic resonance

NP Normal phase

RLCCC Rotational locular counter current chromatography

RP Reverse phase

TLC Thin layer chromatography

viii

UDP-G Uridine diphosphate-D-glucose

UV Ultraviolet

UV-Vis Ultraviolet-visible

VLC Vacuum liquid chromatography

YAN Yangonin

1

Chapter 1

General Introduction

Kava lactones are the biologically active components present in Piper methysticum Forst

(kava plant). Kava lactones are α-pyrones derived through the shikimic acid pathway.

Analysis of the composition of dried kava root shows that it contains up to 20 % kava

lactones. However, the composition can vary between 3-20 % (Lebot et al., 1992). Of the

18 various kava lactones present in the dried roots, 95 % of these consist of six major

compounds namely; desmethoxyyangonin, yangonin, dihydrokavain, kavain,

dihydromethysticin and methysticin (Singh, 1999).

Apart from their existence in free forms, it is believed that a portion of the biologically

active kava lactones exist in bound precursor(s) forms which are inactive. Such precursor

occurrence is seen in many plant natural products. These kava lactone-yielding

precursor(s) release free kava lactones when subjected to various experimental

conditions. It is believed that such a release occurs during the traditional preparation of

kava drink by Tongans and Fijians, where kava is chewed before it is mixed with water.

This instinctive behavior could well lead to the action of salivary enzymes releasing

locked-up kava lactones. Furthermore, it is believed that the presence of stomach acids

within the consumer provides a suitable environment for the release of more kava

lactones from their inactive lactone precursor(s).

2

The wide occurrence of glycosylated secondary metabolites is seen in grapes and wines.

Grape and wine glycosidic precursors are a potential source of latent flavor, which can be

recovered by enzyme and acid hydrolysis (Naiker, 1997). The initial water extract of

dried kava roots is proposed to be composed of kava carbohydrates, organic acids,

proteins, minerals, kava lactones and glycosides. After the removal of all ethanolic

solubles (free kava lactones and other organic compounds medium-to non-polar), the

delactoned residue is believed to be composed mainly of glycosides. The fact that this

research deals with highly polar solvent extracts and with the wide occurrence of

glycosidic precursors in many plant tissues, leads to the postulation that the precursor(s)

of kava lactones are glycosides.

A kava lactone cannot be directly glycosylated because it does not contain any free

hydroxyl group. However it can undergo keto-enol tautomerism prior to glycosylation.

This pattern of glycosylation is very well illustrated with β-damascenone (a potent wine

flavorant) which does not possess any free hydroxyl group, however it undergoes

rearrangement prior to glycosylation (Naiker, 1997).

3

O O

H

MeOB

H B

O O H

OMe

undergoes glycosylation

keto-ester form

enol-ester form

Figure 1.1. Keto-enol tautomerism of kavain prior to glycosylation

Separation of a pure precursor from a pool of numerous naturally occurring glycosidic

precursors is difficult. Such matrix complexity also adds to separation difficulties in any

attempt to perform direct spectrometric analysis (such as MS and NMR) of the individual

precursors, which would provide knowledge of the actual chemical structures. However,

hydrolysis during boiling of these precursors and subsequent analysis of any regenerated

free aglycones could be studied in order to establish precursor occurrence.

Work presented in this thesis explores for the first time the existence of kava lactone-

yielding precursor(s) in Piper methysticum Forst. The existence of precursor(s) for DMY,

YAN and KAV has been established in this research. The detection and quantitation of

these kava lactones, the isolation and characterization of their precursor(s), followed by

aqueous pH reflux and enzymatic hydrolysis of these precursors from roots, is

undertaken.

4

Kava lactone-yielding precursor(s) are a potential source of kava lactones which can be

recovered by enzyme and/or acid hydrolysis. Since kava lactones are the bio-active

ingredients in kava pills and other kava products, identification of these precursors would

enhance the kava lactone content and hence enhance the medicinal value of the plant.

Should the precursor(s) be identified, it would be possible to use them in pills and in

other kava products either directly as a reserve or hydrolyzed and then used.

After this general introductory chapter, the second chapter in this thesis presents an

extensive literature review on the various classes of compounds present in Piper

methysticum Forst. and the wide occurrence of precursors in natural plant tissues.

5

Chapter 2

Literature Review

2.1 The Kava Plant

Piper methysticum Forst. (kava) is a member of the pepper family, Piperaceae. The

family includes about five genera and more than 2000 species of herbs, shrubs, small

trees and woody climbers distributed throughout the tropics, often in rainforests (Lebot et

al., 1992). Piper methysticum is a hardy, slow-growing perennial shrub and can attain

heights of more than three metres. Cultivation of kava is carried out vegetatively using its

rootstock, also referred to as the stump where the active ingredients are located. Piper

methysticum has no rhizomes. Monopodial stems with sympodial branches grow from the

stump. The stump is knotty and sometimes tuberous and often contains holes or cracks

created by partial destruction of the parenchyma (Lebot et al., 1992).

A fringe of lateral roots up to three meters long extends from the rootstock. Kava root

mainly comprises of ligneous fibers and contains more than 60 percent starch. The color

of the rootstock often varies from white to dark yellow depending on the amount of kava

lactone contained in a lemon-yellow resin. The lateral branches emerge from young parts

of the main stems and die and fall away as they age, leaving prominent cicatrices on the

nodes. Foliage in the kava plant is limited and the leaves are thin, single, whole, heart-

shaped, alternate, petiolate, long (8-25cm) and sometimes wider than the length (Lebot et

al., 1992).

6

In early times, the kava plant was distributed eastward through tropical islands by

migrating people, who valued the root both as a drink and medicine. In many islands of

the South Pacific, kava has played an important part in the life of the people, being used

in ceremonial festivals and as a sign of goodwill (Lebot et al., 1992).

2.2 The Bioactive Principle and its Effects

Analysis of the composition of kava rootstock indicates that fresh materials on average

are 80 % water. When dried, rootstock consists of approximately 43.0 % starch, 20 %

fibers, 12 % water, 3.2 % sugars, 3.6 % proteins, 3.2% minerals and 15 % kava lactones.

However, the kava lactone content can vary between 3-20 % depending on the age of the

plant and the cultivar (Lebot et al., 1992)

Kava lactones, sometimes referred to as kava pyrones are the biologically active

ingredients in kava herbal extracts (Hansel, 1968). Kava lactones are very well known

pharmacologically active substances, the details of which and their biological activity can

be found elsewhere (Meyer, 1967; Buckley et al., 1967; Leung and Forster, 1986; Lebot

et al., 1992). Kava lactones, biosynthesized via the shikimic acid pathway are �-pyrones

bearing a methoxy group at carbon 4 and an aromatic styryl moiety at carbon 6. Of the 18

discovered, methysticin, dihydromethysticin, kavain, dihydrokavain,

desmethoxyyangonin and yangonin (Figure 2.1) make up at least 95 % of the kava

lactones present. As reported by Singh (1999), a higher % of kavain and methysticin are

present in free forms compared to other major kava lactones.

7

O O

OCH 3

O

O

(a)

O O

OCH 3

O

O

(b)

O O

OCH 3

(c)

O O

OCH 3

(d)

O O

OCH 3

(e)

O O

OCH 3

H 3CO

(f)

Figure 2.1. Structures of six major kava lactones; methysticin (a), dihydromethysticin

(b), kavain (c), dihydrokavain (d), desmethoxyyangonin (e) and yangonin (f)

The kava drink when consumed due to the presence of kava lactones, provides anti-

anxiety, sedative, anti-insomnia, anti-convulsive, anti-spasmodic, analgesic, anesthetic,

diuretic and diaphoretic properties (Leung and Forster, 1986). Hansel (1968), quoting

Meyer (1965), noted that kava lactones have a potentiating effect on barbituric narcosis.

In an experiment carried out by Meyer on white mice, it was found out that DHM when

injected with hexobarbital sodium caused the animal to sleep for extended hours

compared to injection of only hexobarbital sodium. It was concluded that the potentiating

activity of DHM on barbituric narcosis is particularly pronounced.

8

After comparative tests to measure the analgesic effect of kava, it was found that lower

dosage of DHK and DHM were required for equivalent analgesic effect caused by

acetylsalicylic acid (aspirin). A higher dosage of DHK and DHM compared to morphine

was required for equivalent analgesic effect. Therefore, it was concluded that kava

lactones could have the potential to provide analgesic effect (Lebot et al., 1992).

If fresh kava is prepared by mastication, kava lactones produce local anesthesia of the

chewer’s mouth. It was noted that of the many kava lactones that induce anesthesia,

kavain was particularly effective in surface anesthesia and that the superficial anesthesia

effects of kavain were equivalent to, and last as long as, those of cocaine. Because kava

lactones manifest no toxicity in the tissues, it was preferred over cocaine (Lebot et al.,

1992).

Kava lactones can also act as muscle relaxants. Meyer and Kretzschmar (1965) found that

particularly DHM and DHK are muscular relaxants superior to substances normally used

for such purposes (e.g., propanolol, benzazoles and benzodiazepines). Hansel (1968)

observed that the effects of DHM and DHK on muscles are similar to those of

papaverine. After experiments on frogs, Singh (1983) suggested that kava acts on the

ionic mechanisms that produce muscular contractions. It may be that kava also acts on

the control of muscle relaxation by the central nervous system, as do barbiturates and

tranquilizers.

9

It had also been noted that kava lactones exhibit some form of antimycotic activity after

Hansel (1968) observed that kava extracts had never been attacked by yeasts, bacteria, or

fungi. Although the number of known bactericides is high, substances capable of

stopping the growth of dermatophytic mycoses are rare. Griseofulvine, a substance

commonly used to treat dermatophytic mycoses, has no effect on the strains of

Aspergillus niger, but DHK is the perfect remedy because it completely inhibits the

growth of A. niger (Lebot et al., 1992). It is believed that kava extracts could be used to

prepare orally consumed antimycotics for treatment of diseases such as ringworm and

thrush.

2.3 Other Isolated Compounds from Piper methysticum Forst.

2.3.1 Alkaloids

Alkaloids are compounds of plant origin with complex structures having a nitrogen atom

in a heterocyclic ring. So far over one thousand alkaloids are known, and it is estimated

that they are present in only 10-15 % of all vascular plants. They are found in

cryptogamia, gymnosperms, or monocotyledons. They occur abundantly in certain

dicotyledons. Well-characterized alkaloids have been isolated from roots, seeds, leaves or

bark of plant (Pelletier, 1970). The most commonly occurring piperidine alkaloid in Piper

methysticum so far discovered is pipermethystine.

N

O

O

OAc

Structure of pipermethystine

10

It is found to exist in the leaves and stems of the kava plant (Smith, 1979, 1983; Dragull

et al., 2003). Other piperidine alkaloids found in leaves and stems of Piper methysticum

include awaine and 3�, 4�-epoxy-5�-pipermethysticine (Dragull et al., 2003). The effects

of these piperidine alkaloids on human physiology are unknown and their possible

toxicity on the liver remains to be investigated. However, several pyridone alkaloids with

structures similar to pipermethystine have been shown to be cytotoxic (Duh et al., 1990).

Furthermore, it is believed that pipermethysticine decomposes on standing at room

temperature due to hydrolysis of the amide, to give 3-phenylpropionic acid and the

dihydropyridone (Smith, 1979). 3-Phenylpropionic acid and dihydropyridone exhibit

structural features of 2,5- dihydroxypyridine, which has been shown to affect DNA

integrity in vitro due to its ability to redox cycle (Kim and Novak, 1990, 1991). 3�, 4�-

Epoxy-5�-pipermethysticine is a novel alkaloidal epoxide found in stem peeling of kava.

Epoxidation of certain natural or xenobiotic compounds in liver has been well established

as one of the mechanisms pertinent to hepatotoxicty (Zimmerman, 1999), therefore, this

novel alkaloidal epoxide can be associated with liver hepatotoxicity; however, further

investigation needs to be carried out. Two pyrrolidine alkaloids have also been found to

exist in kava roots as minor components, however, their physiological activity is unclear

(Lebot et al., 1992).

2.3.2 Glutathione

Another compound of interest present in aqueous and 25 % ethanol extracts of kava is

glutathione. Glutathione plays an essential role in the conversion of lactones into

excretable waste products (Schmidt et al., 1999). Increased side effects due to the

11

lactones may occur on glutathione depletion. Glutathione occurs in most cells of the body

in adequate amounts but some individuals show a deficiency linked with cytochrome

P450 (responsible for kava lactone metabolization) (Lomaestro and Malone, 1995). In

these cases, high doses of kava lactones will lead to rapid depletion in glutathione levels

and result in free lactone exposure of the hepatocytes and consequent damage (Zheng et

al., 2000). If however, glutathione supplement is present (as in case of traditional kava

extracts) then this deficiency might be counteracted.

NHNH

OH

O

NH2

OSH

O

O

OH

Structure of glutathione

Other compounds isolated from Piper methysticum in trace quantities and which do not

add much to the activity include flavokavains, an alcohol, a phytosterol, ketones and

organic acids (Lebot and Levesque, 1989). In addition, water-soluble extracts of kava

also contain glucose as the carbohydrate. The extracts when subjected to fermentation,

produced ethanol (Sotheeswaran et al., 1998).

2.4 Occurrence of Polar Precursors in Plant Tissues

So far in the literature, reports have been limited only to the occurrence of free kava

lactones in Piper methysticum. However, as it is believed that a portion of these kava

lactones exist in bound forms and act as precursor(s) of free kava lactones, precursor

12

occurrence in other classes of natural products will also be reviewed. Since the initial

water extract is proposed to be composed of glycosides, review in the following sections

will mainly focus on glycosidically bound precursors. Furthermore, acid and enzymatic

hydrolytic behavior of these naturally occurring precursors will also be reviewed.

2.4.1 Formation and Role of Glycosides

A glycoside is made up of a noncarbohydrate portion (aglycone) attached to the

carbohydrate moiety (glycosyl residue). It is the mixed acetal product resulting from the

exchange of an alkyl or aryl group for the hydrogen atom of the hemiacetal hydroxyl

group of a cyclic aldose or ketose (Courtois et al., 1970). A substance can also be defined

as a glycoside if upon acid hydrolysis it yields one or several monosacchrides and an

aglycone.

The synthesis and hydrolysis of glycosides in living systems can be described by the

general transglycosylation scheme depicted in Figure 2.2 with glucose as an example.

The conjugating agent (generally a glucopyranosyl unit) is attached through an �-

glycosidic linkage to a nucleoside diphosphate (X). Transglycosylation from the

glycosylation agent to the secondary metabolite aglycone Y is always catalyzed by

transferases utilizing nucleotide-activated carbohydrates (NDP-carbohydrate), usually

uridine diphosphate-D-glucose (UDP-G) (Hosel, 1981; Goodwin and Mercer, 1983).

UDP-G is an �-transfer agent which leads to the formation of a �-glucoside.

13

O

OHOH

OH

OH

OX

+ YHO

OHYOH

OH

OH

+ HOX

Figure 2.2. General transglycosylation scheme. X = phosphate, pyrophosphate,

nucleoside diphosphate, carbohydrate, aromatic component, alcohol or hydrogen. Y = a

carbohydrate accepter.

In nature compounds can be glycosylated in two ways. The aglycone portion can directly

bind to the glycone portion if the aglycone carries a free hydroxyl group. This mode of

glycosylation is demonstrated using geraniol as an example (Figure 2.3) (Naiker, 1997).

However, if a free hydroxyl group is absent, the aglycone may be able to undergo

rearrangement to form one prior to glycosylation. This mode of glycosylation is evident

in �-damascenone because �-damascenone itself cannot exist in a directly O-glycosylated

form (Naiker, 1997). It is believed that kava lactones become glycosylated via this mode

since their lactone structures cannot exist in a directly O-glycosylated form. Based on our

postulation that the immediate precursor(s) of kava lactones are β-D-glucopyranosides

which may or may not be further glycosylated, it can be suggested that kavain, via a keto-

enol tautomerism, has an immediate alcohol bearing enol tautomer which is then

glucosylated to give a glucoside. The proposed pathway for the glycosylation of kavain

and its subsequent regeneration (acid and enzymatic) is illustrated in Figure 2.4. The

proposed route for glycosylation of kavain may not be true for desmethoxyyangonin and

yangonin since the lactone ring double bond cannot migrate as proposed. Therefore, it is

postulated that desmethoxyyangonin and yangonin become glycosylated via a different

route. Figure 2.5 illustrates the proposed glycosylation pathway for desmethoxyyangonin.

14

Anthocyanic glycosides have structures similar to the glycosylated form of

desmethoxyyangonin (Naiker, 1997).

CH3 CH3

OH

CH3

O

OH

OH

OHOH

OH

H 2O

CH3CH3

CH3

O+

O

OH OHOH

H

OH

OH

O

OHOH

CH3 CH3

CH3

O

OH

OH

geraniol

beta-D-glucopyranose

geraniol- beta-D-glucopyranoside

Figure 2.3. Glycosylation pathway for geraniol.

15

O O

OCH 3

kavain

O OH

OCH 3

H

H

+

-

glycosylationhydrolysis

O O

OCH 3

O

OHOHOH

OH

beta-D-glucopyranoside of kavain

Figure 2.4. Proposed pathway for the glycosylation/ hydrolysis of kavain

O O

OCH 3

R

O H

OCH 3

O

R

H+

glycosylationhydrolysis

O

OCH 3

O

O HO HOH

O H

O

R

beta-D-glucopyranoside of desmethoxyyangonin/ yangonin

H-

desmethoxyyangonin/ yangonin

R = H: desmethoxyyangonin

R = OCH3: yangonin

Figure 2.5. Proposed pathway for the glycosylation/ hydrolysis of desmethoxyyangonin/yangonin

16

The reasons for the formation of glycosylated compounds in plant tissues is that

glycosylated components are better stored within plant vacuoles and are less reactive

toward other cellular components than the aglycones (Pridham, 1965). Glycosylated

components are often therefore thought of as excretion products or as physiologically

inactive plant storage forms which, under hydrolysis, release the bioactive free aglycones.

As a result glycosylation is an important process in determining the fate of aglycone

secondary metabolites.

Occurrences of glycosidically bound secondary metabolites in plants are observed in

every part of the plant including the leaves (Roscher and Winterhalter, 1993; Ito et al.,

2004), fruits (Adedeji et al., 1991, Adedeji et al., 1992, Fong et al., 1992, Marlatt et al.,

1992) roots and rhizomes (Wu et al., 1991). It has also been found that glycosidically

bound secondary metabolites exist in oak woods and the release of these aglycones

occurs during the process of oak barrel maturation (Spillman et al., 1997, Pollnitz et al.,

2004).

After the discovery of glycosidically bound secondary metabolites in plants, many

speculations and postulations have been made regarding the role of glycosides. Some

important experiments have been undertaken, whose results give supporting evidence for

the hypothetical roles of glycosides in plant tissues. Skopp and Horster (1976) speculated

that glycosidically bound secondary metabolites represent the transport form of the free

aglycones after they discovered that the site of aglycone accumulation was not the same

as the site of production and since the free aglycone cannot penetrate cells without

17

destroying the membranes, the aglycones had to be glycosylated prior to transport. The

role of glycosides as transport derivatives of free aglycones was further supported when a

monoterpene glycoside, which had been applied at the end of the stem of Ocimum

basilicim, was found afterwards in the leaves (Gathen, 1981).

Glycosylation is common in plant tissues, and apart from all the roles already discussed

in this section; it may also function as a protective mechanism and prevent the free

aglycones from destroying membranes. Consequently, it is possible for the free aglycone

constituent of plant tissues to be detected as well as their corresponding glycosides in the

same plant. All these observations can be fitted together and concluded that glycosides

act as reserve sources of their respective free aglycones.

2.4.2 Composition of Glycosidic Precursors in Plant Tissues

As discussed earlier glycosylation changes many properties of secondary metabolites by

transforming them from active constituents to biologically inactive glycoconjugates. As

is observed in grapes this transformation results in loss of fruit flavor which can be

recovered through processing during winemaking and maturation (acid and enzymatic

hydrolysis) (reference). Similarly in Piper methysticum, it is believed that such a release

of free kava lactones from their respective glycosidically bound precursor(s) occurs upon

acid and enzymatic hydrolysis.

18

A common feature of glycosylated secondary metabolites present in plant tissues is that

the components are glucosides in which the glucopyranosyl unit may or may not be

further substituted. It has been often shown in many fruits including grapes, apples,

quince and apricot that further substitution of the glucopyranoside also occurs at C-6. The

second carbohydrate unit of the fruit disaccharide has been identified as �-L-

arabinofuranose, �-L-rhamnopyranose (Williams et al., 1982b), �-D-apiofuranose (Voirin

et al., 1990) in grapes, and �-L-arabinofuranose, �-D-xylopyranose (Schwab and

Schreier, 1990) in apples. Furthermore, gentiobiosides (Guldner and Winterhalter, 1991)

and a trisaccharide glycoside (Herderich et al., 1992) have been reported in quince and

apple fruits respectively.

As reported by Williams et al. (1995), hydrolysis of glycosylated secondary metabolites

therefore, yields an equimolar proportion of aglycones and D-glucose, the latter is termed

the glycosyl glucose (G-G) assay. On the basis of this reasoning, a determination of the

G-G concentration will permit an estimation of the total glycosylated secondary

metabolites present. In order to carry out G-G assay, pure forms of glycosidic precursors

have to be isolated before any conclusive speculations could be postulated about the

structure of the glycosides.

Grape and other fruit glycosidic precursors are potential sources of latent flavor which

can be recovered by enzyme and acid hydrolysis; similarly kava lactone-yielding

precursor(s) are potential sources of the biologically active free kava lactones which can

be recovered by enzyme or acidic hydrolysis. The enhanced kava lactone content is of

19

considerable economic importance to exporters. Knowledge of the chemical nature of

glycosidic precursor(s) would not only benefit, e.g., for wine-making, but also enlighten

the understanding of biochemical and physiological properties of many such plant

metabolites.

2.4.3 Analysis of Intact Glycosidic Precursors

To date, it has been found that many secondary metabolites exist in glycosylated forms;

however, it is only in a few cases that the entire structure of the intact glycoside has been

fully characterized (Ito et al., 2004; Marinos et al., 1992; Sefton et al., 1992; Strauss et

al., 1987: Voirin et al., 1990; Williams et al., 1982a). This discrepancy is caused by the

complexity of the glycosidic isolates obtained from plant tissues and due to the fact that

the individual glycoconjugates of several key compounds are present within this complex

mixture in extremely minute amounts (parts per billion range).

Glycosidic precursors can be directly analyzed using two commonly used analytical

techniques such as GC-MS (Marinos et al., 1992) and HPLC (Fong et al., 1992; Salles et

al., 1988) to provide knowledge about the glycosidic linkages. However, both the

techniques have their own limitations and drawbacks. Marinos et al (1992) used GC-MS

analysis after derivatization to obtain structural information about glycosidic precursors.

This technique has its own drawbacks because derivatization is not applicable in all cases

of polar and thermally labile molecules because of problems with:

20

1. the derivatization technique, i.e. difficulties in preparation, loss in sensitivity,

molecular weight of the precursors may be increased beyond the MS detection

limits.

2. presence of variety of glycosidic precursors in the isolates, therefore limiting

separation and characterization.

3. if the precursors are thermally labile, decomposition may occur prior to analysis.

However, if derivatization of glycosidic precursors is not necessary, then reversed-phase

HPLC can also be employed. Salles et al. (1988) employed an analytical HPLC technique

using a �-cyclodextrin-bound silica gel column to separate glycosidic precursors from

grapes and apricots. Compared to GC-MS, HPLC does not require any volatilization and

therefore, decomposition is not a problem. However, because of the abundance of

glycosidic precursors with various structural complexities of carbohydrate and aglycone

moieties, both of these techniques are not sufficient to fully carry out structural

elucidation prior to some preliminary separation. Thus, preliminary purification using

TLC followed by HPLC was used by Fong et al. (1992) to analyze the limonin 17-�-D-

glucopyranoside (LG) content in fruit tissue of Valencia orange during fruit growth and

maturation.

Due to these separation difficulties, countercurrent chromatography (CCC) in the form of

multilayer coil countercurrent chromatography (MLCCC), droplet countercurrent

chromatography (DCCC) or rotational locular countercurrent chromatography (RLCCC)

of precursor rich fractions is necessary before any structural derivation of glycosidic

21

precursors could be undertaken. Roscher and Winterhalter (1993) demonstrated the use of

MLCCC on glycosidic fractions isolated from Riesling leaves to identify genuine

precursors of the off-flavor-causing compound 1,1,6-trimethyl-1,2-dihydronaphthalene

(TDN). A two-step procedure utilizing preparative MLCCC in combination with

analytical MLCCC yielded two major vine leaf glycosides in pure form, i.e., the �-D-

glucopyranosides of 3-oxo-7,8-dihydro-�-ionol (Blumenol C) and 3-oxo-4,5-dihydro-�-

ionol, respectively. In recent applications of DCCC to research on the precursors of

monoterpenes, norisoprenoids and shikimate derived metabolites of Riesling wine, CCC

partition characteristics and aglycone gas chromatography retention data were used in a

two-dimensional (2D) analysis of the various glycoconjugates (Winterhalter et al.,1990a,

1990b). To achieve this 2D analysis, sequential fraction groups from the DCCC

separation of the intact glycosides were hydrolyzed, and the released aglycones in each

group were separated and characterized using GC-MS.

Combinations of CCC with GC-MS or HPLC and other sophisticated analytical

techniques will answer many questions which are yet open in the field of glycosylated

precursor chemistry.

2.4.4 Analysis of Aglycones after Hydrolysis of Glycosides

As discussed in section 2.3.3, the analyses of intact glycosidic precursors are very

troublesome due to separation difficulties associated with obtaining pure forms of these

precursors. Instead, analysis of the aglycones regenerated after acidic or enzymatic

hydrolysis is preferred. If acidic or enzymatic hydrolysis generates a second crop of

22

aglycones that are similar or biogenetically related to the original aglycone, the presence

of precursor(s) is implied. This approach, mainly developed by Williams et al. (1982c)

and Strauss et al. (1987), has been described as precursor analysis. Caution is necessary

in interpretation of data for the analysis by such experiments, because it does not always

give a full picture of the precursor composition of the product (Sefton, 1991). The major

drawback of such a method is that some glycosidic precursors are not easily hydrolyzed

by available enzymes, while others give artifacts, formed by enzymatic oxidation of the

released aglycones. An additional problem is that some aglycones released after

enzymatic hydrolysis are too unstable to survive GC analyses. These drawbacks have

been illustrated by work on the generation of �-damascenone, a potent flavourant which

can be found free in trace concentrations in grape juices, but which is also formed by acid

hydrolysis of precursor components during bottle ageing (Sefton, 1991).

A similar approach was used by Buttery et al. (1990) and Marlatt et al. (1992) to study

some of the aroma constituents released upon acid and enzymatic hydrolysis of the

glycosidic precursors from tomatoes. It was also found that enzyme-mediated hydrolysis

released only a few volatile compounds in small quantities when compared to acid

hydrolysis (Buttery et al., 1990). Adedeji et al. (1992) also observed similar behavior in

African mango after analyzing the released aglycones using GC from the hydrolysates of

glycosidically bound precursor-rich fractions. A total of 33 compounds was reported in

the glycosidically bound fraction including eight monoterpene alcohols, five aldehydes,

four acids, seven esters and five C13 norisoprenoids.

23

Similar precursor analysis approach as developed by Williams et al. (1982c) and Strauss

et al. (1987) will be used in this investigation to establish precursor(s) existence for kava

lactones upon acid and enzymatic hydrolysis of precursor-rich fractions (partially purified

forms of precursor(s)).

Analysis of the aglycones released upon acid or enzymatic hydrolysis of precursor

isolates provides information that can be used to infer the structural nature of

glycosidically bound constituents that are present in the isolates. Additionally, the

composition of the aglycones released by acid and enzymatic hydrolysis can be compared

with each other and with that of the aglycones present in the original sample. This would

provide an insight into the extent to which the precursors are prone to hydrolysis by the

two modes and whether the free aglycones have any similarities to those that are

glycosylated. The released aglycones can be analyzed using GC or HPLC and further

structural elucidations obtained using MS and NMR.

The major flaw of this approach of precursor analysis is that aglycones are prone to

rearrangement upon release, therefore stability of aglycones is crucial. While the stability

of aglycones is independent of how they are produced, rearrangement patterns and the

chemical nature of aglycones generated from hydrolysis of glycosidic precursors are

strongly influenced by the kinetics of acid catalyzed reaction. Hydrolysis at a pH near

that of the original product could be expected to release aglycones without much

rearrangement or degradation, however, hydrolysis at low pH’s may cause rearrangement

or degradation of the released aglycone. Hydrolysis of precursor concentrates at pH 3.0

24

has been seen to liberate polyols as well as some of their own hydrolysis products, an

outcome which was further enhanced on hydrolysis at pH 1.0 (Williams et al., 1982c)

Another problem with enzymatic hydrolysis is that the products given by hydrolysis of

the precursors depend on the specificity of the enzyme towards the nature of the

carbohydrate conjugating moiety (Gunata et al., 1985). This suggests that the pattern of

aglycones released is very much dependent on the enzyme preparation used for

hydrolysis. To assess this influence a control experiment should be run involving acid

hydrolysis of the aqueous residue remaining after solvent removal of the enzyme

liberated aglycones (Williams et al., 1993). This would provide information on the

presence of glycosides that were resistant to enzyme action.

2.5 Analytical Techniques Involved in Detection and Characterization of Kava

Lactones

Kava lactones, which are the biologically active components present in kava extracts can

be analyzed using various analytical techniques such as HPLC, UV, NMR, IR, TLC, etc.,

however HPLC is the most widely adopted method for kava extract analysis. The other

techniques are often used for characterization of kava lactones.

2.5.1 HPLC Analysis

In a study carried out by Smith (1983), HPLC was used to examine kava lactones from

different parts of kava plant. An alumina (AloxT) column and a mobile phase comprising

of acetonitrile and dichloromethane in a ratio of 1: 90 was used for the purpose of his

25

research. Elution of kava lactones was in the order of KAV, METH, DHK, DHM, with

KAV and YAN peaks not clearly separated (Smith et al., 1983).

In another study carried out by Singh (1999), HPLC was used to detect kava lactones in

kava extracts. A nucleosil column, with a mobile phase of hexane, 1,4-dioxane and

methanol in a ratio of 85: 13: 2 and a flow rate of 1.5 ml/min was employed. Separation

was complete within 24 min and the order of elution of the six major kava lactones was

DMY, DHK, YAN, KAV, DHM and METH. Various other investigations have also used

HPLC analysis to detect kava lactones.

2.5.2 UV Analysis

The total absorbance of a solution at a given wavelength is equal to the sum of

absorbances of the individual components present (Skoog et al., 1997). This relationship

makes possible the quantitative determination of the individual constituents of a mixture,

if their spectra overlap. Furthermore, the high values of the UV extinction coefficients for

kava lactones provide a very convenient method for their quantitative determination.

Young et al. (1966) used UV spectroscopy to characterize kava lactones (DMY, YAN,

DHK, KAV, DHM and METH), after separation using quantitative thin-layer

chromatography. The maximum absorption wavelength (�max) obtained for each kava

lactone was compared with authentic samples to confirm identity.

26

Singh (1999) in his study also used UV spectroscopy to confirm identity of the isolated

kava lactone standards after repeated purification using HPLC prior to separation using

VLC. Similarly, �max values were obtained and compared with authentic samples.

2.5.3 NMR Analysis

NMR spectroscopy is a very useful technique for structural elucidation. This technique

has been employed by Dharmaratne et al. (2002) to determine the structures of various

kava lactones. After obtaining spectroscopic data for the isolated lactone samples in this

present work, and making comparison with data obtained from authentic reference

compounds and with the previous literature, the isolated compound structures were

confirmed.

27

Chapter 3

Isolation and Purity Determination of the Major Kava Lactones

3.1 Introduction

The main difficulty associated with the analysis and quantitation of kava lactones is that

pure standards are very rarely available commercially, and those that are available are

expensive. Therefore, for the quantitative determination of the regenerated kava lactones,

isolation of pure kava lactone standards for the major kava lactones was deemed crucial

for this research.

In this study normal phase analytical HPLC analysis similar to Singh (1999) was used for

the detection of pure kava lactones in the roots of Piper methysticum Forst. Isolation of

the major kava lactones using semi-prep HPLC followed by UV spectroscopy and

1HNMR spectroscopy of the isolated kava lactones for purity confirmation was also

undertaken.

3.2 Results and Discussion

3.2.1 Extraction and Removal of Free Kava Lactones

Water was employed as the initial solvent for the dried root extraction. Even though kava

lactone solubility in water was limited (6 – 8 %, Naiker et al., 2002), of interest to this

research were the highly polar compounds which are believed to be the precursor(s) of

free kava lactones. Hot successive soxhlet extraction with ethanol (200 ml x 5 hrs) was

carried out on the freeze dried aqueous residue for total removal of free kava lactones. It

28

took five soxhlet extractions to obtain a residue totally devoid of free kava lactones

including DMY, YAN and KAV (see methodology section 6.2.3). Chromatogram C

(Figure 3.1) illustrates the chromatogram resulting from the fifth soxhlet extract showing

total removal of free kava lactones. In C, DMY, YAN and KAV, as is observed in the

HPLC chromatogram for the composite standard (chromatogram G, Figure 3.1) and

crude extract (chromatogram A, Figure 3.1), are missing. To further confirm total

removal of free DMY, YAN and KAV, a sixth soxhlet extraction with ethanol was

carried out. The resulting sample showed absence of DMY, YAN and KAV. Thus, it was

apparent that five successive extractions were adequate to achieve a residue totally

devoid of free DMY, YAN and KAV. Furthermore, since the HPLC chromatogram of the

sample resulting from the fifth extract resembled that of the blank injection

(chromatogram B, Figure 3.1), it can be assumed that all other major kava lactones had

also been removed. The resulting delactoned polar residue (18.5 g), after exhaustive

ethanolic extraction, was kept below 0 ºC until analysis.

29

Figure 3.1. HPLC chromatograms obtained for the; crude ethanolic extract (A), blankinjection (B), fifth ethanolic soxhlet extract (C), DMY standard (D), YAN standard (E)and KAV standard (F), composite standard (G).

30

3.2.2 Detection and Isolation of Pure Kava Lactones

After combining the ethanolic extracts resulting from individual soxhlet extraction and

analyzing it using NP analytical HPLC, three different kava lactones, namely DMY,

YAN and KAV (for chromatographic purpose denoted as X, Y and Z respectively) were

detected. The elution order and retention times of the three kava lactones detected in the

ethanolic extract is shown in Table 3a.

Table 3a. Retention times for the three kava lactones

Kava lactone Retention time (min)

DMY 9.48

YAN 13.19

KAV 16.52

The order of elution for these three kava lactones was confirmed through UV and NMR

analysis (discussed in section 3.2.3). Chromatogram A, Figure 3.1 is for the crude

ethanolic extract. In A, peaks for DMY, YAN and KAV are well separated, distinct and

sharp; whereas broad and co-eluted peaks were seen for the other three kava lactones.

Due to these separation difficulties and the low quantities detected for the other three

kava lactones, further isolation and quantitation work was carried out for DMY, YAN

and KAV only. After repeated purification using NP semi-preparative HPLC; 10.8 mg of

DMY, 9.7 mg of YAN and 16.2 mg of KAV were isolated.

31

3.2.3 Purity Determination of Isolated Kava Lactones

The identity of the isolated kava lactones was confirmed by HPLC analysis, UV analysis,

and 1HNMR analysis (see section 6.2.5 of methodology for the various analytical

parameters). From the HPLC results obtained it was determined that DMY, YAN and

KAV was 96%, 87% and 98% pure respectively.

3.2.3a HPLC Analysis

Upon re-injection of the individual kava lactones (DMY, YAN and KAV) and the

composite standard solution of DMY, YAN and KAV, similar retention times (refer

Table 3b) were observed as in the crude extract.

Table 3b. Comparison of retention times for the isolated kava lactones

Kava lactone Crude Extract(min)

Individual injection(min)

Composite Standard(min)

DMY 9.48 9.51 9.53

YAN 13.19 13.15 13.18

KAV 16.52 16.16 16.15

The retention time for DMY, YAN and KAV in the crude extract, individual injection

and composite standard were slightly different due to interaction of different sample

components. The HPLC chromatograms resulting from individual injections for DMY,

YAN and KAV are shown in Figure 3.1 (chromatograms D, E and F respectively). The

chromatographic results suggest that the isolated lactones are quite pure. Furthermore, the

32

HPLC chromatogram obtained for the composite standard solution of DMY, YAN and

KAV (chromatogram G, Figure 3.1) also suggests the presence of only these kava

lactones.

3.2.3b UV Analysis

Figure 3.2. UV spectra of DMY, YAN and KAV.

The observed �max values for the different kava lactones were compared with the reported

values and are shown in Table 3c. Using the observed absorbances for DMY, YAN and

KAV, their respective log � values were calculated and compared with reported values,

which are also shown in Table 3c. The calculated �max values and the log � values for the

three kava lactones deviate slightly from their reported values. Slight deviations in �max

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

220 240 260 280 300 320 340 360 380 400

Wavelength

Ab

sorb

ance

Kavain

Desmethoxyyangonin

Yangonin

33

values may be attributed to the presence of impurities in the sample, and deviation in log

� values may be due to random errors in handling of small volumes of the kava lactone

standards.

Table 3c. Physical constants for kava lactones standards

Observed Literature* Literature**

Standards �max Log � �max Log � �max Log �

DMY 342.84 3.91 342 4.37 342 4.31

YAN 356.72 3.79 355 4.48 357 4.50

KAV 243.87 4.35 244 4.43 245 4.45

* Young et al., 1966

** Singh, 1999

The general structure of kava lactones as illustrated below shows the extent of

conjugation responsible for absorbance at high �max. The extent of conjugation is even

further enhanced in DMY and YAN due to the presence of a C-C double bond between

carbon numbers 5 and 6. This chromophore is responsible for a higher observed �max

value for DMY and YAN compared to KAV in which the double bond between carbon

numbers 5 and 6 is missing.

34

O O

OMe

R 1

R 2

1

2

34

5

67

8

9

1011

12

13

15

14

16

17

General structure for kava lactones

DMY and KAV; R1= R2= H

YAN; R1=OMe, R2=H

DMY and YAN; C=C at C5-C6

3.2.3c 1HNMR Analysis

The interpretation of 300 MHz 1HNMR spectra in CDCl3 for DMY, YAN and KAV is

shown in Table 3d, Table 3e and Table 3f respectively. The structures of DMY, YAN and

KAV are also shown below.

O O

OMe

1

2

34

5

67

8

9

1011

12

1314

15

DMY

35

Table 3d. 1HNMR data for DMY

Observed Literature*

Atom # # of H Ppm # of H Ppm

3 1 5.5 (s) 1 5.44 (d)

5 1 5.9 (t) 1 5.9 (s)

7 1 6.6 (d) 1 6.54 (d)

8 1 7.5-7.6 (m) 1 7.44 (d)

10 1 7.5-7.6 (m) 1 7.48 (m)

11 1 7.3-7.4 (m) 1 7.35-7.31 (m)

12 1 7.3-7.4 (m) 1 7.35-7.31 (m)

13 1 7.3-7.4 (m) 1 7.35-7.31 (m)

14 1 7.5-7.6 (m) 1 7.48 (m)

15 3 3.8 (s) 3 3.76 (s)

Note:

Impurities are seen around 0.5-2.4ppm and 5.3ppm.

36

O O

OMe

MeO

R2

1

2

34

5

67

8

9

1011

12

13

15

14

16

17

YAN

R2 =H

Table 3e. 1HNMR data for YAN

Observed Literature*

Atom # # of H Ppm # of H Ppm

3 1 5.45 (d) 1 5.43 (d)

5 1 5.88 (d) 1 5.86 (d)

7 1 6.4-6.5 (d) 1 6.42 (d)

8 1 7.7 (d) 1 7.42 (d)

10 1 7.4-7.5 (d) 1 7.41 (d)

11 1 6.9 (d) 1 6.87 (d)

13 1 6.9 (d) 1 6.87 (d)

14 1 7.4-7.5 (d) 1 7.41 (d)

15 3 3.8 (s) 3 3.76 (s)

17 3 3.8 (s) 3 3.78 (s)

37

Note:

Impurities seen around 4.1-4.3ppm.

O O

OMe

1

2

34

5

67

8

9

1011

12

13

15

KAV

14

Table 3f. 1HNMR data for KAV

Observed Literature*

Atom # # of H Ppm # of H ppm

3 1 5.2 (s) 1 5.16 (s)

5 2 2.5-2.7 (d, d) 1 2.63 (d,d)

6 1 5.0-5.1 (m) 1 5.02 (m)

7 1 6.2-6.3 (more than d,d) 1 6.23 (d,d)

8 1 6.7-6.8 (d) 1 6.7 (d)

10 1 7.3-7.4 (m) 1 7.25 (m)

11 1 7.3-7.4 (m) 1 7.32 (m)

12 1 7.3-7.4 (m) 1 7.2 (m)

13 1 7.3-7.4 (m) 1 7.32 (m)

14 1 7.3-7.4 (m) 1 7.25 (m)

15 3 3.7 (s) 3 3.72 (s)

38

Note:

Impurities seen around 0.5-2.3ppm, 2.4-2.6ppm and 5.3ppm.

More peaks than d,d due to impurities.

* H. Ranjith W. Dharmaratne et al., 2002

The 1HNMR spectra for DMY, YAN and KAV are attached in the Appendix

39

3.3 Conclusion

Three different major kava lactones were detected and isolated using the methodology

described in this chapter. These were DMY, YAN and KAV. However, chromatographic

peaks seen around these kava lactones which are believed to be those of other major kava

lactones namely DHK, DHM and METH could not be isolated due to the low quantities

of detection and co-elution. For these reasons quantitative work on the regeneration of

kava lactones in the latter part of this research was only carried out for DMY, YAN and

KAV.

The purity determinations of the isolated kava lactones were made on the basis of HPLC

analysis (retention time comparison), UV analysis, and 1HNMR spectroscopy. The UV

and 1HNMR spectra of the isolated standards were very similar to other published data,

showing that the isolated samples contained the required lactones, although still with the

presence of some impurities, as evidenced by the sensitive 1HNMR spectra.

The isolated kava lactone (DMY, YAN and KAV) standards were used in the later

chapters for quantitation of the regenerated kava lactones, therefore precursor(s)

occurrence for only these three kava lactones was established in this research.

The delactoned polar residue obtained after total removal of free kava lactones (DMY,

YAN and KAV) was used in the next two chapters to perform reflux and enzymatic

reactions.

40

Chapter 4

Refluxing at Various Aqueous pH’s and Enzymatic Hydrolysis

of the Lactone Depleted (delactoned) Residue

4.1 Introduction

The delactoned residue was presumably composed of a pool of highly polar compounds

since the initial extraction with water, which is polar. The residue would therefore most

likely contain kava carbohydrates, organic acids, proteins and various glycosides. Due to

the wide occurrence of numerous glycosylated secondary metabolites in nature, it is

believed that these glycosides could act as precursor(s) of kava lactones.

If after the removal of all free kava lactones, the delactoned residue when subjected to

reflux and enzymatic hydrolysis yields a second crop of kava lactones, it could be

inferred that there are precursor(s) of kava lactones in the delactoned residue. These

precursor(s) if present, are the bound or locked-up forms of the highly biologically active

kava lactones.

Reflux of the totally delactoned polar residue under various aqueous pH conditions and

hydrolysis using �-D-glucosidase enzyme on the delactoned residue, is undertaken in this

chapter. Quantitation, of any kava lactones (DMY, YAN and KAV), if regenerated is also

reported.

41

4.2 Results and Discussion

Detection and Quantitation of Regenerated DMY, YAN and KAV Under

Various Aqueous Reflux Conditions and Enzymatic Hydrolysis

When the delactoned polar residue was boiled in water at various pH’s, DMY, YAN and

KAV were found in all the tested pH conditions (chromatograms A-G, Figure 4.1).

Confirmation on the presence of DMY, YAN and KAV in the extracts was done on the

basis of co-injection using their respective standards. DMY, YAN and KAV also

appeared in the hydrolysates upon enzymatic hydrolysis at pH 5 using �-D-glucosidase

(chromatogram H, Figure 4.1).

In contrast, refluxing of the totally delactoned residue with n-hexane, ethyl acetate and

methanol released no detectable levels of free kava lactones. This indicates that

regeneration is only occurring in aqueous medium and is also largely independent of any

solvent change such as that from ethanolic soxhlet extraction to water refluxing of the

polar residue and that the release of free kava lactones is genuine and not merely due to

solvent change. Furthermore, it was also confirmed that regeneration was most efficient

under reflux i.e. heat conditions compared to room temperature or at 0-4 ˚C. It was also

interesting to note that hot water at pH 7 gave little hydrolysis (chromatogram E).

42

Figure 4.1. HPLC chromatograms obtained for reflux at pH 1.0 (A), pH 2.0 (B), pH 4.0

(C), pH 5.0 (D), pH 7.0 (E), pH 7.2 (F) ,pH 8.0 (G) and enzymatic hydrolysis (H) and

control (I).

43

The amount (μg) of DMY, YAN and KAV regenerated under various reflux conditions

and enzyme hydrolysis was quantified using the chromatographic peak area of the

regenerated kava lactones and the average DRF for the individual kava lactone standards.

Section 6.3.4 of Methodology gives the steps involved in obtaining the average DRF for

the three kava lactones.

A sample calculation of DRF for DMY at 0.01mg/ml:

DRF = Amount injected/ Peak area

Amount injected = 0.01mg … (in 1ml)

= 2 x 10-4mg … (in 20�L … injection volume)Therefore:

DRF = 2 x 10-4mg/ 283757

= 7.05 x 10-10

Table 4a shows the peak areas of the three kava lactones in the composite standard at

various dilutions and their DRF values.

44

Table 4a. Peak Area and DRF for DMY, YAN and KAV Standards

Dilution DMY YAN KAV

Stock 1mg/

ml

Peak

Area

DRF

(x10e-10)

Peak Area DRF

(x 10e-10)

Peak Area DRF

(x 10e-11)

0.01 mg/mL 283757 7.05 208977 9.57 3261242 6.13

0.02 mg/mL 801890 4.99 707203 5.66 6428845 6.22

0.04 mg/mL 1343682 5.95 1315789 6.08 10906424 7.34

Av. DRF - 5.99 - 7.10 - 6.56

Using the amount of DMY, YAN and KAV regenerated from the subsequent reflux or

enzymatic hydrolysis, percent of each lactone that could be regenerated from 1 kg of

powdered kava was quantified and is shown in Figure 4.2. A sample calculation for % of

DMY regenerated per kg of powdered kava at pH 1.0 is shown below.

% DMY: Average DRF = Amount injected/ Peak area

5.99 x 10-10 = Amount injected/ 745977

Amount injected = 4.77 x 10-4mg … (in 20μL)

Amount present in 500μL (total volume of sample) = 0.011mg …1.5g of

delactoned residue (used for hydrolysis), therefore amount of DMY present in

92.5g of delactoned residue (mass of delactoned residue obtained from 1kg of

powdered kava) = 0.678mg/kg = 678μg/kg

45

Similarly:

YAN = 2713μg/kg

KAV = 15μg/kg

Therefore % of DMY regenerated per kg of powdered kava =

Amount of DMY/ (Amount of DMY + YAN +KAV) x 100%

% DMY = 678/ (678 + 2713 + 15) x 100%

= 19.9

Similarly:

% YAN = 79.7

% KAV = 0.4

0

10

20

30

40

50

60

70

80

90

pH1.0

pH2.0

pH4.0

pH5.0

pH7.0

pH7.2

pH8.0

Enzym

e

%D

MY

,Y

AN

and

KA

Vre

gen

erat

ed

DMY

YAN

KAV

Figure 4.2. Percent of DMY, YAN and KAV regenerated under various reflux conditions

and enzymatic hydrolysis of the totally delactoned kava residues.

.

46

As is evident from Figure 4.2, the percentage of DMY, YAN and KAV regenerated are

different for different reflux conditions. As shown in Figure 4.2, % regeneration of KAV

is minimum in all the corresponding extracts. However, % regeneration of DMY and

YAN alters. Also of interest to note is that distilled water (pH 7.2) and buffered distilled

water (pH 7.0) show regeneration of DMY, YAN and KAV and that the % regenerated is

very similar.

Since all the seven various pH adjusted solutions contain the same quantity of delactoned

material (1.5 g), the % of regenerated DMY, YAN and KAV might be foreseen to be the

same. However, as seen in Figure 4.2 this was not the case. This is a novel finding which

suggests the possible existence of precursor(s) for DMY, YAN and KAV. The re-

occurrence of DMY, YAN and KAV in the hydrolysate after specific �-D-glucosidase

enzymatic hydrolysis on the delactoned residue, which was nominated to be rich in

glycosides, suggests that the precursors of these kava lactones are indeed glycosides. The

control experiment set without any added �-D-glucosidase enzyme showed absence of

these kava lactones. This suggests that the delactoned residue had no traces of any natural

plant enzymes that could have caused the release of free kava lactones from their

precursors even without artificial acid, base or enzyme catalysis.

47

4.3 Conclusion

The re-occurrence of DMY, YAN and KAV after the removal of all free kava lactones

suggests the existence of kava lactone-yielding precursor(s) for DMY, YAN and KAV.

Reflux behavior of various pH-regulated solutions in this study could also be linked to

the different pH regulated conditions in the human digestive system. It is believed that

such a release of locked up kava lactones as is observed in this study occurs in the human

digestive system after kava ingestion.

The enzymatic hydrolysis results further support the existence of precursor(s) of kava

lactones specifically, DMY, YAN and KAV, and that these precursors are glycosylated.

Such behavior, as is observed in this study, very well explains the importance of the

traditional method of kava drink preparation by Tongans and Fijians. Since saliva

contains hydrolytic enzymes, chewing of kava prior to mixing with water may enhance

the concentration of kava lactones in the traditional beverage through the release of

locked up kava lactones from their precursor(s).

The next chapter will focus on attempts at identifying pure forms of precursor(s) and

subjecting them to reflux for quantifying any amount of DMY, YAN and KAV

regenerated.

48

Chapter 5

Fractionation and Aqueous pH Reflux of the Lactone Depleted Residue

5.1 Introduction

This chapter deals with the fractionation of the delactoned residue using semi-prep RP

HPLC with a view to obtain chromatographically distinguishable precursor-rich fractions

(5 min interval). TLC and analytical HPLC of the delactoned residue is undertaken first

to determine the analytical parameters for semi-preparative HPLC analysis.

Each resulting fraction was subjected to reflux at pH 5 (pH at which overall maximum

regeneration occurs) and the resulting hydrolysate was analyzed using NP analytical

HPLC with a view to quantify the amount of regenerated kava lactones (procedure

termed as precursor analysis, after Williams et al., 1982). For each semi-preparative

HPLC fraction that showed regeneration of kava lactones, the ratio of DMY: YAN: KAV

was determined and compared with the ratio obtained for the other fractions. Thus, if all

fractions have the same type and/ or amount of kava lactone yielding precursor(s), the

respective ratios in each fraction would have to be the same. However, fractions that

show acceptable difference would suggest that these fractions have chromatographically

distinguishable precursor(s) or groups of precursors.

Each precursor rich fraction was also subjected to 1HNMR analysis to obtain information

on the structure, type and nature of the precursors.

49

5.2 Results and Discussion

5.2.1 Thin Layer Chromatographic Analysis of the Delactoned Residue

Upon TLC analysis of the delactoned residue, best separation was observed with a

solvent system of acetonitrile and water in a ratio of 9: 1. It was not possible to obtain

distinct spots; however, long streaks were seen. Other solvent systems that did show

some separation were acetonitrile: methanol, methanol: water and acetonitrile: water:

methanol in various compositions.

5.2.2 Reverse Phase Analytical HPLC Analysis of the Delactoned Residue

Best separation was achieved at a flow rate of 0.5 mL/min. Detection was performed at a

variety of wavelengths ranging from 200-400nm. Strongest chromatographic detection

was observed at 210nm (range where phenolic and other mono glycosides absorb).

Acetonitrile: methanol (80: 20 and 90: 10) used as the mobile phase gave good separation

but column blockage was a problem which resulted in build up of extremely high pump

pressure. This made analysis difficult since column damage and pump damage could

have occurred. A similar problem was encountered when a solvent system of acetonitrile:

water (90: 10, 85: 15, 80: 20 or 75: 25) was used. A three solvent system of acetonitrile:

water: methanol in various ratios was used next. A lot of irregular peaks over a long

period of time were seen. High pump pressure build up was also encountered and

therefore, this solvent system was not used. Since it was believed that the delactoned

residue is mainly composed of highly polar components and that complete elution was a

problem (column blockage and excess build up of pump pressure), it was concluded that

50

a highly polar solvent system was necessary to overcome this. Therefore, the water

composition in the mobile phase with either acetonitrile or methanol was enhanced. It

was observed that acetonitrile and water in a ratio of 1: 3 at flow rate of 0.5 mL/min and

detection at 210nm gave a relatively better separation without much backpressure

problems. These parameters were then used for semi-preparative analysis.

5.2.3 Reverse Phase Fractionation and Aqueous pH Reflux of the Delactoned

Residue

Figure 5.1 shows the RP semi- preparative HPLC chromatogram of the delactoned

residue, obtained using the parameters developed from TLC and analytical HPLC which

was fractionated at five minute intervals over 30 minutes.

Figure 5.1. RP HPLC chromatogram for the delactoned residue

RP semi-preparative fractionation of 2 g of delactoned residue over a run time of 30

minutes (five minute collection interval) resulted in collection of 7.02 mg of fraction 1,

10.14 mg of fraction 2, 11.3 mg of fraction 3, 38.80 mg of fraction 4, 9.30 mg of fraction

5 and 15.8 mg of fraction 6. Upon subjecting each fraction to aqueous reflux at pH 5 and

51

analyzing the resulting hydrolysates using NP analytical HPLC, the following

chromatograms were obtained (Figure 5.2).

Figure 5.2. HPLC chromatograms of pH 5 reflux for fraction 1 (A), fraction 2 (B),

fraction 3 (C), fraction 4 (D), fraction 5 (E) and fraction 6 (F).

52

5.2.4 Precursor Assay

The peak areas of the regenerated DMY, YAN and KAV and their respective av. DRF of

their standards were used to determine the percent of each kava lactone regenerated per

kg of powdered kava and is shown in Figure 5.3. A sample calculation for % of DMY

regenerated per kg of powdered kava in fraction 1 is also shown below.

% DMY: Average DRF = Amount injected/ Peak area

5.99 x 10-10 = Amount injected/ 691488

Amount injected = 4.14 x 10-4mg … (in 20μL)

Amount present in 349μL (total volume of sample) = 7.23 x 10-3mg present in

2.0g of delactoned residue (used to obtain fractions), therefore amount of DMY

present in 92.5g of delactoned residue (mass of delactoned residue obtained

from 1kg of powdered kava) = 0.334mg/kg = 334μg/kg

Similarly:

YAN = 414μg/kg

KAV = 137μg/kg

Therefore % of DMY regenerated per kg of powdered kava =

Amount of DMY/ (Amount of DMY + YAN +KAV) x 100%

% DMY = 334/ (334 + 414 + 137) x 100%

= 37.7

Similarly:

% YAN = 46.8

% KAV = 15.5

53

Note that the volume used to re-dissolve the dichloromethane extracts (contain released

kava lactones) of the fraction hydrolysates varied due to the different masses of fractions

used for hydrolysis. Fraction 1, 2, 3, 4, 5 and 6 used 349μL, 333μL, 383μL, 513μL,

271μL and 276μL respectively.

0

20

40

60

80

100

120

1 2 3 4 5 6

Precursor fraction

%D

MY

,Y

AN

and

KA

Vre

gen

erat

ed

DMY

YAN

KAV

Figure 5.3. Percent of DMY, YAN and KAV regenerated at pH 5 reflux for the precursor

fractions.

Maximum % of regeneration of DMY, YAN and KAV is seen in fractions 5, 3 and 1

respectively, whereas minimum % regeneration of DMY, YAN and KAV is seen in

fractions 1, 5 and 5 respectively. Fractions 1, 2 and 3 are early eluting polar fractions and

have shown similar patterns of regeneration.

54

Furthermore, the ratios of regenerated kava lactones in each precursor fraction were

determined (Table 5a). The ratios provide information on the quantities of kava lactone-

yielding precursors for each kava lactone relative to the other in each fraction.

Table 5a. The ratios of DMY, YAN and KAV regenerated (μg) per kg of powdered kava

Precursor fraction DMY:YAN:KAV

1 2:2:1

2 30:30:1

3 50:60:1

4 150:40:1

5 3810:1:15

6 120: 3:1

Fractions 1-3 have similar ratios. This suggests those fractions may be composed of very

components. Low quantities of KAV regeneration are seen in all the fractions except in

fraction 5. It is therefore assumed that low quantities of KAV exist in precursor form

since its existence in free form as reported by Singh (1999) is high relative to DMY and

YAN. The quantities of DMY and YAN regenerated in most of the fractions are high

relative to KAV. This suggests the existence of high quantities DMY and YAN in

precursor forms rather than in free forms. Singh (1999) also reported low values of free

DMY and YAN in kava samples. Furthermore, as observed in Table 5a, quantities of

55

DMY and YAN regeneration are very similar in a number of the fractions. This supports

the proposed pathway for glycosylation of DMY and YAN as discussed in chapter 2.

The ratio of regenerated DMY, YAN and KAV in each precursor fraction is different

from the other fractions. This suggests the existence of chromatographically

distinguishable precursors for each kava lactone in each fraction that maybe structurally

and chemically different.

5.2.5 1HNMR Analysis of Precursor Rich Fractions

Using the 1HNMR spectra of fractions (see Appendix) it was not possible to deduce the

structures of the precursors, however the spectra indicated the presence of various

functionalities including:

� anomeric protons at 4.94 ppm and 5.34 ppm (fractions 3 and 4), 4.94 ppm and 5.1

ppm (fractions 5 and 6).

� resonance resulting from the presence of sugars around 3.0-4.0 ppm in all the

fractions.

� methyl singlets and triplet around 1.1-1.3 ppm in all the fractions.

� large water peak at 4.66 ppm resulting from the exchange of the sugar protons with

deuterium of the D2O, as well as residual H20 in the D2O and possibly in the sample.

� DCM peak at 5.41 ppm, which was used to clean the tubes.

56

These combined results suggest that the precursors may be glycosides; however

confirmation of the actual structures was not possible due to the fact that pure precursors

were not isolated. The presence of large water and DCM peaks also made structural

elucidation more difficult. Of interest to note is that the spectra do not show the presence

aromatic lactone-moiety protons. The presence may be masked by the sugar protons due

to relatively high sugar concentration in the fractions. The presence of aromatic signals

may also not be detected since the sample solutions appear quite dilute.

57

5.3 Conclusion

Using the procedure outlined in this chapter, it has not been possible to separate pure

forms of kava lactone yielding precursors due to separation difficulties such as pressure

problems and matrix effects in the delactoned residue. Direct quantitation of pure

precursors could not therefore be undertaken. However, partially pure forms of

chromatographically distinguishable precursor-rich fractions were isolated, their reflux

behavior studied, and the regenerated kava lactones quantified.

RP semi-preparative fractionation of delactoned residue resulted in six precursor-rich

fractions, all of which yielded a different ratio of DMY, YAN and KAV. This infers that

each precursor-rich fraction is composed of at least one precursor for each kava lactone

and the hence existence of multiple precursors that are chemically/structurally different.

High field 1HNMR data of the fractions suggests the presence glycosides which supports

the enzyme hydrolysis data. However, actual structures were not deduced due to the

complexity of the fractions. Each precursor-rich fraction consists of components of

different polarities based on the elution rate. A common feature of glycosylated

secondary metabolites present in many plant tissues is that the components are glycosides

which may or may not be further substituted to form a di/tri or polysaccharide whose

polarities will be different. Based on this fact and that each precursor-rich fraction is of

different polarity to the other, there is a genuine possibility that the precursors of DMY,

YAN and KAV may be further glycosylated.

58

5.4 Outlook

Further purification of the partially purified groups of precursors would require the highly

efficient countercurrent chromatography (CCC) in forms of rotational locular

countercurrent chromatography (RLCCC) and droplet countercurrent chromatography

(DCCC) in order to obtain pure precursors of kava lactones. All fully purified precursors

would finally be subjected to LC-MS and NMR spectroscopy techniques to facilitate

structural identification.

59

Chapter 6

Methodology

6.1 General

All solvents were of high purity at purchase and redistilled before use. Laboratory

research grade water was used.

All NP analytical HLPC analyses were carried out at 2.0 mL/min with a mobile phase

comprising of n-hexane: ethyl acetate (6: 4). A Waters HPLC system comprising of a

model 515 pump, Rheodyne injector fitted with a 20 μL loop, connected to an Econosil

Silica column (250 x 4.6 mm, 10 μm particle size) and a Waters 2487 dual λ absorbance

detector, was used. Data were recorded and integrated using a Waters 746 data module

recorder. Analysis was monitored at 254 nm and at an attenuation of 256.

All NP semi-preparative HPLC analyses were carried out at 1.5 mL/min with the same

mobile phase and Waters HPLC system, except for the Rheodyne injector fitted a with

200 μL loop and a μ-Porasil column (300 x 7.8 mm, 10 �m particle size).

All RP semi-preparative HPLC analyses were carried out at 0.5 mL/min with a mobile

phase comprising of acetonitrile: water (1: 3). The same Waters HPLC system as for

normal phase analysis was used. An Econosil C18 column (250 x 10mm, 10�m particle

size) was employed. Analysis was monitored at 210 nm at an attenuation of 256.

60

All UV analyses were carried out using Perkin Elmer’s Lambda 16 UV/VIS

spectrophotometer accompanying Perkin Elmer computerized spectroscopy software. The

absorbance of the kava lactone standards was measured in a quartz cell (1 cm path

length) in the range of 200-400 nm. HPLC grade methanol was used as the solvent for

UV analysis.

All NMR analyses were carried by Dr David Tucker of University of New England,

Australia. A Bruker Avance 300 MHz spectrometer using a 5 mm inverse 1H/BB probe

with z-gradient was used.

Dynavac Engineering FD3 freeze drier was used to freeze dry samples where necessary.

BUCHI Rotavapor R-114 equipped with BUCHI Waterbath B-480 was used for rota-

evaporation.

All solvents and samples were membrane filtered (0.45 μm cellulose acetate filter) and

ultrasonically degassed prior to HPLC analysis. Samples were flushed with nitrogen prior

to analysis.

HANNA Instruments 8521 pH meter was used for adjustment of pH.

61

6.2 Isolation and Purity Determination of the Major Kava Lactones

6.2.1 Sample

Dried powdered rootstock (4 kg) of Piper methysticum Forst. was obtained from a local

kava dealer at $25 per kg. The approximate age of this Piper methysticum Forst. sample

was 4 years and was cultivated locally.

6.2.2 Aqueous Extraction

The dried powdered rootstock (200 g) was soaked in 1 L of water and left overnight at

room temperature. The aqueous extract was magnetically stirred prior to filtration using

cheesecloth. The resulting filtrate was freeze dried to obtain a residue (25 g) which was

kept below 0 °C until required for further analysis.

6.2.3 Removal of Free Kava Lactones

The freeze dried solid residue (25 g) was subjected to five rigorous hot successive soxhlet

extractions with ethanol (200 ml, 5 hrs) to achieve a sample totally devoid of free kava

lactones (18.5g). Confirmation of this was achieved by analyzing the resulting crude

extract of each successive ethanolic extraction using NP analytical HPLC (see section

3.2.1). Extraction was ceased once no traces of kava lactones were seen. To confirm total

removal, a sixth soxhlet extraction was carried out. The totally delactoned residue was

kept below 0 °C until required for further analysis.

62

6.2.4 Detection and Isolation of Pure Kava Lactones

The resulting crude ethanolic extracts were combined and concentrated to dryness under

vacuum (water bath temp. 28 °C). The dried residue was redissolved in a suitable HPLC

mobile phase (n-hexane: ethyl acetate; 6: 4) to make a stock solution of crude kava

lactone extract. NP analytical HPLC was used for detection of the major kava lactones.

Blank injections (mobile phase) were made before and between each analysis. NP semi -

preparative HPLC analysis was used for the isolation of the major kava lactones.

6.2.5 Purity Determination of Isolated Kava Lactones

HPLC, UV and 1HNMR analysis were used for purity determination of the isolated kava

lactone standards. An estimate on the level of purity of the isolated kava lactones was

made on the basis of the percentage peak area for each kava lactone from the HPLC

chromatograms obtained for individual injections (see section 3.2.3).

A composite standard solution of the isolated kava lactones was made in a suitable HPLC

mobile phase and analyzed using NP analytical HPLC. The retention times of the isolated

kava lactones were compared with those of the kava lactones present in the crude kava

lactone sample. Individual isolated kava lactone standard solutions were also made and

analysed using NP analytical HPLC and their retention times compared with the kava

lactones present in the crude kava lactone extract and the composite standard solution. To

further confirm the identity of the isolated standards, co-injection (individual kava

lactone) was also done.

63

The isolated kava lactones were also subjected to UV spectroscopic analysis to confirm

identity. Absorbance at maximum wavelength (λmax) was obtained and compared with

literature values. Using the absorbance at λmax, molar absorptivities for the individual

kava lactones were calculated and compared with the literature values.

Each isolated kava lactone was also subjected to 1HNMR analysis at 300MHz for

conformation of identity against the reported literature (Dharmaratne et al., 2002).

1HNMR spectra were run in CDCl3 at 30 degrees C. 32 scans were collected for each

kava lactone (see Spectra 1-3, Appendix).

6.3 Refluxing at Various Aqueous pH’s and Enzymatic Hydrolysis of the

Delactoned Residue

6.3.1 Reflux of the Delactoned Residue at Various Aqueous pH’s

The delactoned residue (1.5 g portion) was dissolved in distilled water (200 ml), and the

process repeated to obtain seven solutions. The pH’s of these solutions were separately

adjusted to 1.00, 2.00, 4.00, 5.00, 7.00 (buffered), 7.23 (distilled water only) and 8.00

respectively, and each mixture refluxed for 1.5 hrs. The pH’s of these solutions were

obtained using a pH meter, which was calibrated before each reading. For adjustment to

obtain a pH of 1.00, 2.00, 4.00 and 5.00, 0.10 M HCl was used, and to adjust to a pH of

8.00, 0.10 M NaOH was used. A pH 7.00 buffer of 0.10 M NaOH and 0.10 M KHP was

used. The resulting solutions from subsequent refluxes were cooled to room temperature

and neutralized with HCl or NaOH.

64

6.3.2 Enzymatic Hydrolysis of the Delactoned Residue

Citric/phosphate buffer (pH 5.0) was prepared by mixing 48 ml of aq. citric acid solution

(A) (0.1 M) and 53 ml of aq. di-sodium hydrogen orthophosphate solution (B) (0.2 M).

The pH was adjusted as required by addition of small amounts of solution (A) and

solution (B). 0.5 g of delactoned residue was dissolved in 5 ml of pH 5.0 buffer. A

solution of almond emulsin (a β-glucosidase enzyme) (2 mg) was made up in pH 5.0

buffer (1.0 ml) and 0.5 ml (1.0 mg) of this solution was added to the sample. The mixture

was incubated at 35 ± 2 °C for 6 hrs, then cooled to room temperature. Five drops of

saturated NaCl solution were added to overcome emulsion problems. A control was set

without any enzyme added to the delactoned sample.

6.3.3 Extraction of Regenerated Kava Lactones

Each cooled, neutralized solution resulting from subsequent aqueous pH reflux or

enzymatic hydrolysis was magnetically stirred with dichloromethane (100 mL, 3 hrs).

The resulting organic layer was separated and then dried with anhydrous sodium sulfate

prior to concentrating it to dryness under vacuum (water bath temperature; 28 °C). The

dried organic extract was redissolved in a suitable HPLC mobile phase (3 mL, 6: 4 n-

hexane: ethyl acetate) and concentrated to 500 μL under a stream of nitrogen gas prior to

HPLC analysis.

6.3.4 HPLC Analysis of Regenerated Kava Lactones

Each organic extract was subjected to NP analytical HPLC analysis. Triplicate injections

were made for each organic extract. Quantitations of the regenerated kava lactones from

65

their subsequent refluxes were done on the basis of the peak areas of the regenerated kava

lactones and the average detector response factors (DRF) for the individual isolated kava

lactone standards. A series of dilutions (0.04 mg /mL, 0.02 mg/mL and 0.01 mg/mL) of

the composite stock standard (1 mg/L) were made and analysed using NP analytical

HPLC. Using the formula;

DRF = Amount injected (mg): the DRF for each kava lactone was calculated for eachPeak Area

dilution and the average DRF calculated for each kava lactone. The average DRF for

each kava lactone was then used to quantify the amount of each kava lactone regenerated

from its subsequent refluxes.

6.4 Fractionation and Aqueous pH Reflux of the Delactoned Residue

6.4.1 Development of Analytical Parameters for Reverse Phase HPLC Analysis of

the Delactoned Residue

6.4.1.1 Thin Layer Chromatographic Analysis

TLC of the delactoned residue was necessary to identify the best condition for semi –

preparative (fractionation) HPLC analysis. TLC of the totally delactoned residue was

carried out on silica gel GF254 glass plates. Acetonitrile, methanol and water were the

solvents used in various compositions as the mobile phase for TLC analysis (comprising

of components of high polarity). A UV lamp was used to visualize the developed plates.

66

6.4.1.2 Reverse Phase Analytical HPLC Analysis

A Waters HPLC system comprising of 1525 binary HPLC pump, 717 plus autosampler,

2996 photodiode array detector aided with Empower software was used for analytical

HPLC analysis. A C18 Econosil column (250 x 4.6 mm, 10 μm particle size) was used for

this analysis. A two solvent system and a three solvent system comprising of acetonitrile,

methanol and water in various compositions was used as the mobile phase. The

delactoned residue was dissolved in the appropriate mobile phase and in water for

injection. Analysis was carried out at different flow rates for each mobile phase.

Chromatograms were scanned over a range of wavelengths (200–400 nm) to determine a

specific wavelength at which maximum chromatographically distinguishable compounds

could be detected.

6.4.2 Reverse Phase Semi-preparative HPLC Analysis and Fractionation

After determination of suitable HPLC analytical parameters (mobile phase; 1:3,

acetonitrile: water, flow rate of 0.5 mL/min, water as the solvent to dissolve the

delactoned residue, wavelength of detection - 210nm and an attenuation of 256) for

reverse phase analysis of the delactoned residue, semi-preparative HPLC was used for

fractionation of the delactoned residue. Individual fractions (five minute collection

interval) were collected over a period of 30 minutes. This resulted in collection of 7.02

mg of fraction 1, 10.14 mg of fraction 2, 11.3 mg of fraction 3, 38.8 mg of fraction 4,

9.30 mg of fraction 5 and 15.80 mg of fraction 6.

67

6.4.3 Aqueous pH Reflux of Individual Reverse Phase Fractions

The fractions collected in section 6.4.2 were subjected to aqueous reflux at pH 5 (pH at

which maximum regeneration of kava lactones occurs). The amount of kava lactone

regenerated in each fraction under aqueous pH 5 reflux was quantified as in section 6.3.4.

6.4.4 1HNMR Analysis of Individual Reverse Phase Fractions

Fractions 3-6 resulting from semi-preparative fractionation were subjected to 1HNMR

analysis at 300 MHz. Spectra were run in D2O at 30 degrees C. 128 scans were collected

for the individual fractions (see Spectra 4-7; Appendix).

68

Chapter 7

References

Adedeji, J., Hartman T. G., Rosen, R. T., Ho, C. T. (1991). Free and glycosidically bound

aroma compounds in hog plum (Spondias mombins L.). J. Agric. Food Chem., vol. 39,

1494-1497.

Adedeji, J., Hartman T. G., Lech, J., Ho, C. T. (1992). Characterization of glycosidically

bound aroma compounds in the African mango (Mangifera indica L.) J. Agric. Food

Chem., vol. 40, 659-661.

Buckley, J. P., Furgiuele, A. R., O’Hara, M.J. (1967). Pharmacology of Kava 2.

Psychopharmacology Bulletin 4 (3), 11-12.

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Appendix

1. 1HNMR Spectra of DMY

76

2. 1HNMR Spectra of YAN

77

3. 1HNMR Spectra of Kavain

78

4. 1HNMR Spectra of Semi-preparative Fraction 3

79

5. 1HNMR Spectra of Semi-preparative Fraction 4

80

6. 1HNMR Spectra of Semi-preparative Fraction 5

81

7. 1HNMR Spectra of Semi-preparative Fraction 6

82