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POTENTIAL
APPLICATION OF KAVA
(Piper methysticum F.)
IN NEMATODE CONTROL
A thesis submitted in partial fulfillment of the
requirements for the degree of
Master of Science
By
Ranjeeta D. Singh (BSc, PGDip)
School of Biological, Chemical and Environmental Sciences
Faculty of Science and Technology
The University of the South Pacific
November 2006
Potential Application of Kava (Piper methysticum F.) in Nematode Control
DECLARATION
I declare that this submission is a result of my own investigation and has not been
submitted in any form for another degree or diploma at any university. To the best of my
knowledge, it contains no material written by another author or previously reported,
except where due acknowledgement is credited in the text.
Candidate: RANJEETA D. SINGH
The current research was conducted under our supervision and we are certain that this isthe sole work of Ms Ranjeeta D. Singh.
Supervisors:
Dr Mani Naiker
Chemistry Supervisor
School of Chemical Sciences
Faculty of Science and Technology
University of the South Pacific
Dr Uma Khurma
Biology Supervisor
School of Biological Sciences
Faculty of Science and Technology
University of the South Pacific
ii
Potential Application of Kava (Piper methysticum F.) in Nematode Control
ABSTRACT
Kava (Piper methysticum) has long history of its use in social and ceremonial gatherings
and for medicinal purposes by the Pacific islanders. Research has identified the active
ingredients as the kava lactones, which have been widely tested for various bioactivity
properties. The water soluble compounds on the other hand have not been scientifically
explored much. Work presented in this thesis reports for the first time the nematicidal
property of polar extracts of dried kava roots.
The water soluble compounds of dried kava root material referred to as sample X and the
highly polar extract, sample Y (sample X less the ethanol solubles) has been tested for
juvenile mortality and suppression of egg hatching. Samples Y1, Y2, Y3 and Y4 obtained
by semi preparative HPLC separation of sample Y, were tested for juvenile mortality
only. The results of these experiments were compared with the activity of gallic acid as
the standard. Efficacy of kava powder was tested in pot experiments as soil amendment.
Samples X, Y, Y1, Y2, Y3, and Y4 showed significant activity towards juvenile mortality
and suppression of egg hatching. Samples X of FJ and VA kava showed some differences
in activity towards juvenile mortality but similar activity was noted for egg hatching
experiments for both FJ and VA kava samples. H-NMR analyses of the four fractions
isolated have shown the possibility of the presence of glucoside like compounds in the
polar extract of kava. Kava powder when mixed with soil significantly decreased the root
iii
Potential Application of Kava (Piper methysticum F.) in Nematode Control
galls on tomato plants. Allowing kava to degrade in the soil before planting seedlings
minimized the phytotoxic effect while being effective in controlling nematodes.
These results have indicated that kava does have the nematicidal polar compounds.
Further purification of the extracts obtained will lead to the isolation of individual
compounds. GC-MS and NMR analysis of individual compounds will further provide
information on the structure of these compounds.
iv
Potential Application of Kava (Piper methysticum F.) in Nematode Control
ACKNOWLEDGEMENTS
I gratefully take this opportunity to thank my supervisors Dr Mani Naiker and Dr Uma
Khurma (School of Biological, Chemical and Environmental Sciences) for their support,
guidance and criticism during my research.
Special thanks to Dr David Tucker of University of New England, Armidale, NSW,
Australia for his valuable time in performing NMR analysis.
The University Research Committee and the School of Pure and Applied Science
Research Committee are acknowledged for providing the necessary funds for the
operating costs of this research. The Scholarship Committee is duly thanked for awarding
me the Graduate Assistantship.
I am highly indebted to Dr Mathias Schmidt (HERB Research, Germany), Professor
Yadhu Singh (South Dakota State University, USA) and Dr Linton Winder, (School of
Biological, Chemical and Environmental Sciences, USP) for helping with obtaining
journal papers. The assistance by Dr Linton Winder and Dr Badru Doza of the University
of the South Pacific in the statistical analysis of the results is also highly appreciated.
The technicians of School of Biological, Chemical and Environmental Sciences
especially Mr Vas Deo, Steve, Ragni and Dinesh are acknowledged for their help in
locating the necessities for this project. Praneel, Roneel and Abhinesh are acknowledged
Potential Application of Kava (Piper methysticum F.) in Nematode Control
for their support in carrying the experiments. Sincere thanks to Sachin and Kirti for their
advice and guidance for the required instrumentation of this project.
I offer my heartfelt thanks to my parents (Mr & Mrs Vijay K. Singh), my brothers (Jagen
and Rakesh) and my sister (Geeta) for their patience, encouragement and contribution to
my education.
Finally my special thanks to my friends Kirti, Sachin, Modi, Ranjani, Vikashni,
Riteshma, Sunny, Babeeta, Pedro and Lawrence for helping with the writeup, giving
enthusiasm and keeping me company when I needed them most.
vi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
PUBLICATIONS
Part of the work described in this thesis has been presented as:
1. Singh, R., Naiker, M. and Khurma, U. R. 2004. Evaluation of Nematode Suppressive
Potential of Kava. International Kava Conference, Suva, Fiji.
2. Khurma, U.R. and Singh, R. 2005. Effect of Kava as Soil additive on Root-knot
Nematode Population. ONTA 2005, Organization of Nematologists of Tropical
America at Viña del Mar, CHILE.
vi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
LIST OF ABBREVIATIONS
BOD
CCC
cm
cv.
DCCC
DCM
FJ
ft
g
g/mL
GC-MS
H-NMR
HPLC
hrs
in.
J1
J2
L
LC-MS
m
min
biological oxygen demand
countercurrent chromatography
centimeters
cultivar
droplet countercurrent chromatography
dichloromethane
Fiji
feet
grams
grams per millilitre
Gas chromatography coupled with mass spectroscopy
proton nuclear magnetic resonance spectroscopy
High Performance Liquid Chromatography
hours
inches
first stage juveniles
second stage juveniles
litres
liquid chromatography coupled with mass spectroscopy
metres
minutes
vi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
mL
mL/min
mL/kg
mm
nm
NMR
ppm
PPN
RLCCC
RKN
semi-prep
sp.
TLC
UV-Vis
VA
w/v
w/w
X-VA
X-FJ (stored)
X-FJ (fresh)
Y-FJ
milliliters
milliliters per minute
milliliters per kilogram
millimeters
nanometers
nuclear magnetic resonance spectroscopy
part per million
plant parasitic nematodes
rotational locular countercurrent chromatography
root knot nematodes
semi preparative
Species
Thin layer chromatography
Ultra-violet visual
Vanuatu
weight by volume
weight by weight
Freeze dried aqueous residue of Vanuatu Kava
Freeze dried aqueous residue of Fiji Kava Stored at 4 °C for six
months
Freeze dried aqueous residue of Fiji Kava, freshly prepared
Freeze dried aqueous residue of Fiji Kava minus ethanol
extracts
ix
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Y-VA Freeze dried aqueous residue of Fiji Kava minus ethanol
extracts
YI-FJ First eluted fraction collected from Y-FJ in semi-preparative
HPLC
Y1-VA First eluted fraction collected from Y-VA in semi-preparative
HPLC
Y2-FJ Second eluted fraction collected from Y-FJ in semi-preparative
HPLC
Y2-VA Second eluted fraction collected from Y-VA in semi-preparative
HPLC
Y3-FJ Third eluted fraction collected from Y-FJ in semi-preparative
HPLC
Y3-VA Third eluted fraction collected from Y-VA in semi-preparative
HPLC
Y4-FJ Fourth eluted fraction collected from Y-FJ in semi-preparative
HPLC
Y4-VA Fourth eluted fraction collected from Y-VA in semi-preparative
HPLC
uL microlitres
um micrometres
Potential Application of Kava (Piper methysticum F.) in Nematode Control
LIST OF FIGURES
Figure 1.1 Structures of six major kava lactones 4
Figure 1.2 Structure of Pipermethystine 5
Figure 1.3 Structures of pigment molecules 6
Figure 1.4 Structures of cinamic acid, pinostrobin and 5,7-dimethoxyflavone 7
Figure 1.5 Structures of phenolic compounds identified in kava…………………......8
Figure 1.6 Structure of 2-furfuraldehyde 23
Figure 1.7 Structure of 20-hydroxyecdysone 24
Figure 1.8 Structures of benzaldehyde, citral, furfural, menthol and
a-terpineol 24
Figure 2.1 HPLC chromatogram showing peaks of the ethanol solubles…………….34
Figure 2.2 HPLC chromatogram of the final ethanol extract from soxhlet…………..34
Figure 2.3 HPLC chromatogram for sample Y showing the groups of
peaks which were isolated 35
Figure 3.1 Tomato roots infected by RKN as viewed with naked eyes………………40
Figure 3.2 Live juveniles in motion in water as viewed under 40x magnification…...45
Figure 3.3 Dead juveniles viewed under 40x magnification (note the slightly
curved to almost straight posture) 46
Figure 3.4 Kava powder mixed in soil and left for degrading in the plot land……....50
Figure 3.5 The author planting the tomato plants in the pots after inoculating
with juvenile nematodes 50
xi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Figure 3.6 Juvenile Mortality for FJ kava fractions 63
Figure 3.7 Juvenile Mortality for VA kava fraction 63
Figure 3.8 Treatment of egg masses with sample X-FJ (fresh)………………………67
Figure 3.9 Treatment of egg masses with sample X-VA…………………………......67
Figure 3.10 Treatment of egg masses with sample Y-FJ 69
Figure 3.11 Treatment of egg masses with sample Y-VA 69
Figure 3.12 Comparison of activity of samples X of FJ and VA kava
with Gallic Acid for egg hatching 73
Figure 3.13 Comparison of activity of samples Y of FJ and VA kava
with Gallic Acid for egg hatching 73
Figure 3.14 Zero weeks degradation FJ kava 75
Figure 3.15 Zero weeks degradation VA kava 75
Figure 3.16 Two weeks degradation FJ Kava 78
Figure 3.17 Two weeks degradation VA kava 78
Figure 3.18 Four weeks degradation FJ kava 79
Figure 3.19 Four weeks degradation VA kava 79
Figure 3.20 Six weeks degradation FJ kava 80
Figure 3.21 Six weeks degradation VA Kava 80
Figure 3.22 Root gall numbers against kava concentration for FJ kava………………83
Figure 3.23 Root gall numbers against kava concentration for VA kava…………….83
xi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
LIST OF TABLES
Table 3.1 FJ kava extracts tested for juvenile mortality at room
temperature 54
Table 3.2 Analysis of Variance Results for FJ Kava Samples
tested at room temperature 54
Table 3.3 FJ kava extracts tested for juvenile mortality at 28°C…………………......57
Table 3.4 Analysis of Variance Results for FJ Kava Samples tested at 28 °C…….....57
Table 3.5 VA kava extracts tested for juvenile mortality at 28°C……………………59
Table 3.6 Analysis of Variance Results for VA Kava Samples tested at 28 °C……...59
Table 3.7 Comparison of samples X of FJ and VA kava with gallic acid……………60
Table 3.8 Comparison of samples Y of FJ and VA kava with gallic acid……………61
Table 3.9 Analysis of Variance Results for FJ and VA Kava fractions tested
at 28°C 62
Table 3.10 Results for Sample X-FJ (fresh) 66
Table 3.11 Results for Sample X-VA 66
Table 3.12 Results for Sample Y-FJ 68
Table 3.13 Results for Sample Y-VA 68
Table 3.14 Observed plant heights for the various treatment experiments
of EJ and VA kava 81
Table 3.15 Observed root lengths for the various treatment experiments
ofFJ andVAkava 81
xi
Potential Application of Kava (Piper methysticum F.) in Nematode Control
TABLE OF CONTENTS
Declaration ii
Abstract iii
Acknowledgements v
Publications vii
List of Abbreviations viii
List of Figures xi
List of Tables xiii
1.0 Introduction 1
1.1 Kava 1
1.2 Chemistry of Kava 3
1.3 Biological Activity of Kava 9
1.4 Toxic effects of other Piper spp 13
1.5 Nematodes 15
1.6 Effect of Meloidogyne spp on Plants 16
1.7 Nematode Management 18
1.7.1 Nematicides for Nematode Control 18
1.7.2 Natural products for Nematode Control 19
1.7.3 Compounds Isolated from Plants with
Nematicidal Activity 22
1.8 Aims and Objectives of Present Study 25
2.0 Kava Extractions and Separation 26
2.1 Introduction 26
2.2 Methodology 27
2.2.1 General Methodology 27
2.2.2 Aqueous Extraction 28
2.2.3 Removal of Ethanol Solubles 28
2.2.3.1 HPLC analysis of Sample Y to verify the
removal of Ethanol Solubles 29
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.2.4 Separation of Sample Y 29
2.2.4.1 Identification of x max 30
2.2.4.2 Mobile Phase Composition Determination for
separation of Sample Y using Thin Layer
Chromatography 30
2.2.4.3 Gradient HPLC Analysis of Sample Y……………... 31
2.2.4.4 HPLC Separation of Potential Nematicidal
Compounds from Sample Y 32
2.2.5 1H-NMR Analysis of Fractions of Sample Y…………………..33
2.3 Results and Discussion 34
2.3.1 HPLC analysis of Sample Y to verify the
removal of Ethanol Solubles 34
2.3.2 1H-NMR Analysis of Fractions of Samples
Y1,Y2, Y3and Y4 35
2.4 Conclusion 37
3.0 Bioassays 38
3.1 Introduction 38
3.2 Methodology 40
3.2.1 General Methodology 40
3.2.2 Nematode Culturing and Species Identification……………... 41
3.2.3 Sample Preparation 42
3.2.4 Laboratory Assays of Kava Extracts 43
3.2.4.1 Juvenile Mortality Tests 44
3.2.4.2 Egg Hatching Experiments 46
3.2.4.3 Comparison with standard (Gallic Acid)……………. 47
3.2.5 Pot Experiments (Soil Amendment Experiments)……………..48
3.2.5.1 Preparation of Pots and Soil for Experiments………...48
3.2.5.2 Experimental Setup 48
3.2.5.3 Planting of Seedlings and Inoculating………………..49
3.2.5.4 Plant Observations 51
3.3 Results and Discussion 52
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.1 Laboratory Assays of Kava Extracts 52
3.3.1.1 Juvenile Mortality Experiments 52
a) FJ Kava Extracts Tested at Room
Temperature 52
b) FJ Kava Extracts Tested at 28 °C 55
c) VA Kava Extracts Tested at 28 °C………………...57
d) Comparison of sample X of FJ
and VA Kava with the standard 59
e) Comparison of sample Y of FJ and VA
Kava with the Standard 60
3.3.1.2 Juvenile Mortality Experiments of the
Fractions 61
3.3.1.3 Egg Hatch Experiments 64
a) Activity of samples X and Y of FJ
and VA kava 64
b) Comparison of Activity of Samples X
and Y of FJ and VA Kava with Standard……….....70
3.3.2 Pot Experiments (Soil Amendment Experiments)
for FJ and VA Kava 74
3.3.2.1 No Degradation Allowed (0 weeks)……………….....74
3.3.2.2 Two Weeks of Degradation 76
3.3.2.3 Four Weeks of Degradation 76
3.3.2.4 Six Weeks of Degradation 77
3.3.2.5 Comparison of Observed Results
at Different Concentrations over various
Degradation Periods 82
3.3.2.6 General Discussion 84
3.4 Conclusion 87
4.0 General Conclusions 89
5.0 Reference 91
Appendices 105
Potential Application of Kava (Piper methysticum F.) in Nematode Control
CHAPTER 1
1.0 INTRODUCTION
1.1 Kava
Piper methysticum Forst is a tropical shrub that grows throughout the Pacific Islands. It
belongs to the pepper family (Piperaceae) and is also known as kava, asava pepper, or
intoxicating pepper. It grows to an average height of 6 ft (1.83 m) and has large heart-
shaped leaves that can grow to 10 in. (25.4 cm) wide. This plant is cultivated for
commercial and social purposes and is harvested after 3 to 5 years from cultivation
(Davis and Brown 1999; Walji 1998). The roots and lower parts of the plant are dried,
pounded and mixed with water to make an intoxicating drink that serves as a traditional
beverage in Fiji and the South Pacific countries.
Various cultivars of kava are found in the Pacific. Fiji has eleven distinct cultivars while
Vanuatu, believed to be the origin of kava, has eighty-two different cultivars (Lebot and
Le'vesque 1989). Farmers distinguish them on the basis of morphological and ancillary
characteristics, physiological effects on drinkers, and the region where the cultivar was
thought to have originated (Walji 1998).
Kava drink is believed to help break social barriers, settle interpersonal conflicts and
enhance social ties. The drink is prepared by mixing ground or masticated roots and stem
Potential Application of Kava (Piper methysticum F.) in Nematode Control
bases with water. Usually it is served in a half coconut shell, and the total contents are
always drained in one draught (Davis and Brown 1999). The Pacific Islanders also offer
it to gods, spirits and ancestors as a sign of respect, to obtain favor and to appease their
resentment and anger if due respect has not been shown to them and to communicate with
the supernatural world.
Various authors have described the effect of the drink differently. Some stated the thirst
relieving potential of kava due to its pleasant, cooling, aromatic and numbing effect on
the mucous membrane of the tongue (Singh 1986; Walji 1998). Te Rangi Hiroa a
Polynesian from New Zealand, who often drank kava found it cooling, refreshing, and
stimulating without being intoxicating. Other reports describe it with bitterness and
burning taste in the mouth but the first effect is a numbing and astringent effect on the
tongue and the inner lining of the mouth (Singh 1986; Walji 1998).
Apart from being used as a beverage, kava is also used for medicinal reasons by the
Islanders. Gout, rheumatism, diarrhea, asthma, venereal diseases and convulsive
disorders were some of the conditions, which were treated with kava (Duve 1976; Lebot
et al., 1992, 1997; Singh and Blumenthal 1992). A tea made from the roots of kava is
used as a diuretic for kidney and bladder ailments in Fiji. Coughs, colds and sore throats
are treated with kava as well. A decoction of the root given to mothers who had given
births is known to prevent them from getting pregnant again while leaves are chewed as a
contraceptive measure. Wounds are known to be treated with juice extracted from fresh
leaves (Forster 2000; Lebot et al, 1992, 1997).
Potential Application of Kava (Piper methysticum F.) in Nematode Control
1.2 Chemistry of Kava
The medicinal uses of kava had initiated research into the chemistry and biological
activity of kava. Many active compounds have been isolated from kava and the majority
of the biological activity has been accredited to the kava lactones. Kava lactones are
documented to have pharmacological properties such as anticonvulsive, antiepileptic,
antifungal and local anesthetic effects (Cambie and Ash 1994; Cordell 1998).
Chemical analysis of the kava root stock has shown that fresh roots contain on average 80
% water, but when dried, consists of approximately 43 % starch, 20 % fibers, 12 % water,
3.2 % sugars, 3.6 % proteins, 3.2 % minerals, and 20 % kava lactones (can vary between
3 % to 20 % depending on the age of plant and the cultivar) (Lebot et al, 1992, 1997;
Singh 1986; Singh and Blumenthal 1992).
The kava lactones are a group of α - pyrones with a methoxy group at carbon 4 and an
aromatic styryl moiety at carbon 6. Eighteen different lactones have been identified in
kava of which the six major ones include kavain (1), methysticin (2), yangonin (3), 7,8-
dihydrokavain (4), 7,8-dihydromethysticin (5) and desmethoxyyangonin (6). The lactones
are lipid like compounds contained within oil cells and thus would be deemed to be
generally insoluble in water, therefore the traditional preparation of kava drink produces
an emulsion of the lactones (Basko 2002; Lebot et al., 1992, 1997; Walji 1998).
Potential Application of Kava (Piper methysticum F.) in Nematode Control
CH3
,ChU
Figure 1.1: Structures of six major kava lactones
A novel pyridone alkaloid, pipermethystine (7) has also been identified in P.
methysticum. This alkaloid was not present in the roots but formed a minor component of
the stem and leaves (Cambie et al., 1997; Smith 1979, 1983; Dragull et al., 2003).
Pipermethysticine decomposes on standing at room temperature due to hydrolysis of the
amide, to give 3-phenylpropionic acid and the dihydropyridone (Smith 1979). Other
piperidine alkaloids found in leaves and stems of Piper methysticum include awaine and
3a, 4a-epoxy-5P-pipermethysticine (Dragull et al., 2003). N-cinnamoylpyrrolidine and N-
Potential Application of Kava (Piper methysticum F.) in Nematode Control
[m-methoxycinnamoyl]-pyrrolidine were three other alkaloids isolated from the roots
(Sotheeswaran 1987).
Alkaloids are compounds of plant origin with complex structures having a nitrogen atom
in the heterocyclic ring. So far over 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 plants
(Pelletier 1970).
CH
Figure 1.2: Structure of Pipermethystine
Glutathione is another compound of interest found in aqueous and 25 % ethanol extract
of kava. Glutathione is soluble in high polarity solvents and the concentration of it
increases with increasing polarity of the solvents. This compound has been found to be
reacting with kava lactones. The decolourisation of total kava lactone solution in
presence of glutathione supports this interaction. The loss of colour is due to the opening
of the lactone ring (Whitton et al., 2003). The interaction is similar to those reported for
Potential Application of Kava (Piper methysticum F.) in Nematode Control
sesquiterpene lactones with glutathione. Such reaction of glutathione is important in
converting the kava lactones into excretable waste products (Schmidt et al., 1999).
Other compounds isolated from kava are flavokawins, an alcohol, a phytosterol, ketones
and organic acids (Lebot and Levesque 1989). Pigment materials, flavokawin A (8) and
B (9) were isolated and reported in 1963 and have been associated with the skin
discoloration in chronic kava drinkers (Singh 1986).
H3C
Figure 1.3: Structures of pigment molecules
Analysis of water-soluble components of kava indicated the presence of glucose as the
carbohydrate in addition to the pharmacologically active lactones. This was determined
through thin-layer and paper chromatographic techniques (Sotheeswaran et al., 1998).
Treatment of the delactoned residue of kava (after the lactones and other ethanol solubles
were removed) with ß-D-glucosidase showed regeneration of some of the lactones
(Naiker et al., 2006). Thus indicating the presence of glucosidic compounds together with
carbohydrates in the highly polar water extract.
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Extraction of powdered kava root by steam followed by lyophilization produced the most
pharmacologically active water-soluble component. Analysis of this fraction revealed
presence of aldehydes and ketones but no nitrogen was detected (O'hara et al., 1965).
Hot water and methanol extraction of kava lactones revealed the presence of some other
compounds of interest. The methanol extract yielded bornyl esters of 3,4-methylenedioxy
cinnamic acid, cinnamic acid (10), pinostrobin (11), flavokawain B(9) and 5,7-
dimethoxyflavonone (12). The aqueous extract contained the previously reported kava
lactones upon TLC analysis (Wu et al, 2002).
OH
OH O CH
Figure 1.4: Structures of cinnamic acid, pinostrobin and 5,7-dimethoxyflavonone
Potential Application of Kava (Piper methysticum F.) in Nematode Control
O O
HOOH OH
HO "y" HO
OH 13 OH 14
O
HO
OH
15 HO
O
OH
OH 18
19
Figure 1.5: Structures of phenolic compounds identified in kava
Potential Application of Kava (Piper methysticum F.) in Nematode Control
High Performance Liquid Chromatography (HPLC) analysis of 80 % methanolic extract
of kava revealed the presence of phenolic compounds. These were identified to be gallic
acid (13), protocatechuic acid (14), p-hydrobenzoic acid (15), p-coumaric acid (16),
ferulic acid (17), salicylic acid (18) and trans-o-coumaric (19). Trans-cinamic acid (10)
was also reported together with these phenolic compounds (Xuan et al, 2003 ).
Elemental composition of the dried kava powder includes N: 0.37, P: 0.27, K: 0.63, Mg:
0.07 and Ca: 0.46 % (Xuan et al. 20031).
1.3 Biological Activity of Kava
The different lactones present in kava are said to be the psychoactive ingredients, which
act as sedatives, soporifics, analgesics, anticonvulsives, local anesthetics, muscle relaxant
and diuretics (Davis and Brown 1999; Walji 1998). Strong activity against fungi is also
reported for these kava lactones (Duve 1976; Lebot et al., 1992; Singh and Blumenthal
1992). Minor skin infections are usually treated with kava in highlands of Indonesia and
in Papua and New Guinea (Davis and Brown 1999).
Kava resin which contains the lactones is reported to have a weak sleep inducing action,
paralyzing effect on sensory nerves and stimulating result on the smooth muscles before
paralyzing them (Walji 1998). It was also noted that there was an increase in the activity
of kava when mixed with saliva. Saliva reduces the starch component thereby increasing
the concentration of the kava lactones (Walji 1998). The action of saliva can be compared
with that of acid hydrolysis whereby kava lactones are regenerated from a totally devoid
Potential Application of Kava (Piper methysticum F.) in Nematode Control
sample (Naiker and Prasad 2005). The regeneration was explained as their release from
precursors, which were postulated to be glucosides. The amylase enzyme in saliva could
be behaving in a similar way thus increasing the concentration of kava lactones.
The water-soluble fractions of the steam distillate of the kava showed a depression of the
spontaneous motor activity in albino mice without any effect on forced motor activity (O'
hara et al., 1965). Kava resin as well as the water soluble, delactoned extracts of kava
reduced amphetamine-induced hyper motility in mice (Duffield et al., 1989). The
aqueous extract of kava, containing methysticin and dihydromethysticin showed
enhanced spasmolytic activity (Sotheeswaran 1987). Tumor induced by okadaic acid on
mouse was inhibited by methanol extract of kava as well as that of green tea
(Sotheeswaran 2002). This indicated that kava had the potential to prevent humans from
tumors as effectively as green tea, which has well-known anticancer activity.
The ethanolic extract of kava produced dose-dependent anxiolytic-like behavioral
changes and reduction in locomotor activity in mice. Flumazenil, a competitive
benzodiazepine receptor antagonist was found to block the anxiolytic and sedative effects
of diazepam but it did not have any effect on the behavioral actions of kava (Garrett et
al., 2003). This indicates that the anxiolytic-like behavioral changes and sedation by kava
is not mediated through the benzodiazepine-binding site on the GABA-A receptor
complex.
10
Potential Application of Kava (Piper methysticum F.) in Nematode Control
There have not been any reports of any kind of toxic effects of kava among the Pacific
Islanders. The only detrimental effect noticed is the 'kava dermopathy' which is
commonly known as 'kani' and is visible in heavy kava drinkers. In this condition the
skin gets dry and is covered with scales. It is inferred that flavokawain is responsible for
this condition. Flavokawain interferes with the normal uptake and metabolism of some of
the B-group vitamins (Sotheeswaran 1987). Ruze (1990) carried out an examination on
over 200 Tongans and came to the conclusion that the dermopathy was not due to the
deficiency of niacin, as was the case for acquired ichthyosis. Postulations on interference
of compounds present in kava with cholesterol metabolism have been made for this skin
condition as well (Norton and Ruze 1994).
Kava has been linked to gastrointestinal complications in heavy consumers but there is
limited scientific evidence for this (Malani 2002). Poor health has been associated with
heavy kava drinkers (Mathews et al., 1988). This is mainly due to poor eating habits of
these individuals. Other conditions associated with kava drinking are impaired vision,
enlargement of pupils, disturbances in occulomotor equilibrium, worsening of
Parkinson's syndrome and interactions with central nervous system depressants
especially benzodiazepines (Singh 2005).
Several pyridone alkaloids with structures similar to pipermethystine have been shown to
be cytotoxic (Duh et al., 1990). The compounds 3-phenylpropionic acid and
dihydropyridone exhibit structural features of 2,5- dihydroxypyridine, which has been
shown to affect DNA integrity (Kim and Novak, 1990, 1991).
11
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Kava root extracts in 70 % methanol has been found to inhibit the growth of following
five fungi; Fusarium solani, Pyricularia grisea, Rhizopus stolonifer, Taphrina deformans
and Thanatephorus cucumeris. The highest effect was seen on R. stolonifer, where 100 %
inhibition was observed (Xuan et al., 20031). It inhibited mycoses (fungal infection) of
the skin, which is generally very resistant to treatment (Hansel and Klaproth 1966). The
kava pyrones have fungistatic properties against a wide range of fungi including many
which are pathogenic to humans (Singh 1986; Sotheeswaran 1987). Dihydrokavain (kava
lactone) was effective against Aspergillus niger (Hansel and Klaproth 1966).
Antibacterial properties of P. methysticum against Gonococcus sp. (the cause for
gonorrhea), Colon bacillus and Salmonella typhi have been reported as well (Walji
1998). The crude lactones showed antimicrobial properties against a number of bacteria
including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,
Acinetobacter baumii, Salmonella typhi and Candida albicans (Lal 2003).
The kava lactone yangonin, is reported to have amoebicidal properties. In vitro tests
against Entamoeba histolytica resulted in 100 % elimination after 48 hours of incubation.
This activity was comparable to that of many commercial drugs like enteroviaform,
enteroquinol, clefamide and furamidazole (Sonfi et al., 1983). Another amoeba against
which 80 % ethanol extract of kava had detrimental effect was Acanthamoeba castellani
(Whitton et al., 2003). It was noticed that this extract had 100 % inhibition of amoeba but
the inhibition was reduced to 40 % when the ethanol extract was mixed with glutathione.
12
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Some herbicidal properties of kava have also been reported (Xuan et al., 20031). A strong
inhibition of barnyard grass, monochoria, and knot grass was noted when kava was
applied to soil. Eight phenolic compounds have been identified in kava which have also
been isolated from many allelopathic plants (Xuan et al., 20032).
1.4 Toxic effects of other Piper spp.
Insect defense properties are common in the plants of the family Piperaceae, including
the common spice black pepper, Piper nig rum L. and around 1000 other tropical species.
Research has shown that the plants of the Piper genus contain over 200 secondary
compounds. The amides present are responsible for providing the 'hot pungent' taste and
the biological activity to this genus. The most active piperamide discovered is pipericide
(Dyer et al, 2004).
The piperamides belong to the unsaturated isobutylamides which is a well known group
of insecticidal compounds found in various Piper species. Various amides including
pipericide have been isolated from the fruits, stem and leaves of P. nig rum, P.
acutisleginum, P. khasiana, P. longum, P. pedicellosum and P. thomsoni. Having
neurotoxic effect, this group of compounds has a knockdown as well as lethal action
against insects which are resistant to pyrethroid insecticides (Parmar et al,. 1997). Piper
extracts have the ability to anaesthetize adult and larvae of potato beetle. This activity on
the beetle population was due to piperamides (Scott et al., 2003). Aqueous extract of
Piper colubrinum had nematicidal effect on root knot nematodes. Two crystalline
13
Potential Application of Kava (Piper methysticum F.) in Nematode Control
compounds identified in this plant with nematicidal properties were 5,3'-dihydroxy-7-
methoxy flavone and 5,3',4'-trihydroxy-7-methoxy flavone (Eapen and Kumar 2005).
In vitro testing of whole plant of Piper longum L. showed fungicidal activity against
phytopathogenic fungi namely Pyricularia grisea, Rhizoctonia solani, Botrytis cineria,
Phytophthora infestans, Puccina recondite and Erysiphe graminis. Hexane extract
contained piperoctadecalidine, a piperidine alkaloid which had a powerful antifungal
activity against Puccina recondite (Park et al., 2003).
Crude volatile oil and its petroleum ether and dichloromethane extracts of Piper betle L.
showed insecticidal and fungicidal effects on some pests of cotton (Solsoloy et al., 2001).
The crude oil controlled sucking insects such as Aphis gossypii and Amrasca bigutulla
and acted as ovicide against Helicoverpa armigera and Pectinophora gossypiella. The
two solvent fractions repressed growth of certain fungi (Sclerotium rolfsil, Fusarium
oxysporum and Rhizoctonia solani) in laboratory experiments. The fractions mainly
contained monoterpenes and sesquiterpenes. The crude oil was generally more effective
than the two fractions.
Piper sudangrass (Lb) is a summer crop and grows on hot, fertile and irrigated soil. It is
harvested for hay for horses and cattle. It is known to reduce populations of many species
of nematodes and symphylans when grown as a rotation crop. As it decomposes, it
releases cyanide from its roots, which has nematicidal properties (Chitwood 2002).
14
Potential Application of Kava (Piper methysticum F.) in Nematode Control
1.5 Nematodes
Nematodes are tiny worm like, unsegmented organisms. They play an important but
unrecognized role in soil fertility and agricultural productivity. Many of the nematodes
are free living found in the oceans, freshwater habitats and in soils. A small group of
these are parasitic. Plant parasitic nematodes (PPN) make about 20 % of the species
within the phylum Nematoda (Ferraz and Brown 2002). They live in soil where they
infect plant roots and underground tissues. These nematodes are estimated to cause an
annual crop loss of more than 10 %, amounting to billions of US dollars (Sasser 1989;
Shurtleff and Averre III 2000; Ferraz and Brown 2002) and root-knot nematodes (RKN)
are responsible for major share of this damage. Sandy soil is usually preferred by PPN
since this allows the larvae to move freely from their hatching site to new roots. The
optimum temperature of 30 - 32 °C is necessary for reproduction which completes in
about 15 to 18 days (Ferraz and Brown 2002; Weischer and Brown 2000).
Of more than 2,000 different species of plant parasitic nematodes, the most economically
important groups are the root knot (Meloidogyne spp.), cyst (Heterodera spp.), root lesion
(Pratylenchus spp.), reniform (Rotylenchulus spp.) and sting (Belonolaimus spp.)
nematodes (Shurtleff and AverreIII 2000; Siddiqi 2000; Ferraz and Brown 2002). The
nematodes of interest for bioassays in this investigation are the root knot nematodes
(RKN), the group of greatest agronomic importance. RKN (Meloidogyne spp.) are widely
distributed and have an extensive host range. They form galls on roots that block water
and nutrient flow to the plant, therefore stunting growth, impairing fruit production and
15
Potential Application of Kava (Piper methysticum F.) in Nematode Control
causing foliage to yellow and wilt. This causes the roots to become rough and pimpled
and susceptible to cracking. RKN parasitism adversely affects the yield quantity and
quality. They also leave open wounds thus providing an entry to plant pathogenic fungi
and bacteria.
Root knot nematodes affect crops in the tropical and subtropical regions more severely as
climatic conditions favor growth and reproduction of the damaging species. This genus
comprises of more than 80 different species of which four are unquestionably the most
important plant parasitic nematodes on the planet (Sasser and Carter 1985; Taylor and
Sasser 1978). These four species are M. arenaria, M. hapla, M. incognita and M.
javanica and are distributed widely in agricultural areas around the world. Meloidogyne
incognita (Kofoid and White) Chitwood is the most widespread and damaging of the
RKN species and can parasitize a wide range of hosts (Yepsen 1984; Sasser and Carter
1985; Caswell and Bugg 1991; Siddiqi 2000). The awareness of RKN occurrence in the
Pacific Island countries that are highly dependent on primary production provides a good
reason for this investigation.
1.6 Effect of Root Knot Nematodes on Plants
Sexual dimorphism is quite clear in this genus. Females are obese and saccate. They are
sedentary and are embedded in the roots where they lay eggs while males are typically
vermiform. The first stage juveniles (J1) from embryogenesis moult within the egg and
hatch as second stage juveniles (J2). The motile and infective J2, migrate through the soil
16
Potential Application of Kava (Piper methysticum F.) in Nematode Control
in search of roots of suitable host plants (Croll 1970). The movement of juveniles in the
soil may last for months if environmental conditions are not favorable. The energy they
require at this time comes from fatty reserves accumulated in their intestines.
The second stage juveniles usually penetrate the roots just above the root cap. They move
between undifferentiated root cells and come to rest with their heads in the developing
stele near the region of cell elongation and bodies in the cortex. The cell walls are pierced
with the stylets and the secretions from the esophageal glands are injected. These
secretions lead to the enlargement of cells leading to the formation of giant cells, possible
dissolution of cell walls, enlargement of nuclei and changes in composition of the cell
contents (Ferraz and Brown 2002). The intense multiplication of cells around the larval
head lead to the formation of the distinct galls that are known as knots.
The formation of galls suppresses root elongation and the infected roots appear darkened,
frequently contain longitudinal cracks and many of the fine feeder roots are destroyed.
Nematode infection is also known to disrupt carbohydrate partitioning and affect the
phenol oxidase activity. This makes the plant more susceptible to cold injury and
bacterial infections, which have direct killing effect on the plants (Weischer and Brown
2000; Ferraz and Brown 2002). The overall growth of the plants is greatly reduced
especially in young plants growing in nurseries.
17
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Shoot symptoms observed in infected plants are stunting, wilting and leaf chlorosis.
These observed symptoms indicate the inefficiency of the shortened root system to
uptake water and nutrients from the soil. Root and plant weight reductions up to 50 %
have been noted in several infected crops while mortality in forest nurseries was more
than 50 % (Ferraz and Brown 2002; Sasser 1989).
1.7 Nematode Management
Various nematode management options include use of chemicals, resistant crop varieties,
crop rotation, using organic matter and green manures, growing nematode suppressive
crops, flooding or leaving the soil fallow, solarizing soil, and employing methods of
biocontrol (Oka et al, 2000; Taylor and Sasser 1978). Chemical control has been the
preferred method of nematode control in many countries. Many of the chemicals used to
kill or suppress the pests persist in the environment for long periods. Those having
bioaccumulative properties end up in the human body through the food chain where at
high levels they have detrimental effects (Harris 2000).
1.7.1 Nematicides for Nematode Control
Use of chemicals for nematode control has been reported since 1911 when carbon
bisulfide was used to kill soil nematodes and the left over chloropicrin from the First
World War was used by pineapple growers to control root knot and reniform nematodes
(Sasser and Carter 1985). Later in the 1940s, several halogenated hydrocarbons such as
18
Potential Application of Kava (Piper methysticum F.) in Nematode Control
methyl bromide, a mixture of dichloropropane and dichloropropene, and ethylene
dibromide, was being marketed as nematicides.
These chemicals were efficient fumigant nematicides and had high vapor pressure. When
injected in soil they could disperse by gaseous diffusion through the soil pore spaces and
kill the nematodes by interfering with their cell metabolism (Sasser and Carter 1985;
Ferraz and Brown 2002). Many of these chemicals were banned when information on the
danger of these chemicals was found as soil residues, groundwater contamination and
residues in edible plant produces. Since no alternatives for these chemicals were
available, research was initiated to explore biological control methods which are
environmentally friendly. Natural products from plant and microorganism populations
have been explored and are desired since they are easily degraded in the soil (Prakash and
Rao 1996; Taylor and Sasser 1978).
1.7.2 Natural products for Nematode Control
Many plants have been used as organic amendments in soils for the control of root knot
nematodes. The chemical properties of the plants used as amendments contribute to their
efficacy towards the control of nematodes (Rodriguez-Kabana 1986). Compounds with
nematicidal properties are released when organic matter is decomposed or are
synthesized by microorganisms during the decomposing process. These compounds
include organic acids, hydrogen sulfide, nitrogenous ammonia, phenols, and tannins
(Chavarria-Carvajal et al., 2001). Organic matter in soil also stimulates microbial
19
Potential Application of Kava (Piper methysticum F.) in Nematode Control
populations of fungi and bacteria, which may be foe to nematodes (Morgan-Jones and
Rodriguez-Kabana 1987).
Aqueous extracts of Conyza canadensis, Blumea oblique, Amaranthus viridis, Eclipta
prostrate, Azadirachta indica, Chromolaena odorata, Strychnos nuxvomica and Pimenta
dioica were found to inhibit egg hatching and cause juvenile mortality of RKN in
laboratory experiments (Begum et al., 2003; Eapen and Kumar 2005). Soil amendments
with powdered root material of C. canadensis, B. oblique, A. viridis, E. prostrate in pots
of brinjal plants reduced nematode population and root knot development (Begum et al.,
2003). Incorporation of green leaves of S. nuxvomica in basins of black pepper reduced
the foliage from yellowing due to nematode infections (Eapen and Kumar 2005).
Ethanolic extracts of rhizomes of Artemisia vulgaris inhibited egg hatching upto 50 % at
2.35 mg/ml and caused 50 % mortality among second stage juveniles within 12 hrs of
exposure at 55 mg/ml. The ethanolic extract when applied to soil reduced root galling on
a susceptible host. The extract did not seem to lose activity upon dilution with water
when stored in the dark at 25 °C for up to 15 days (Costa et al., 2003). Hexane extract of
the leaves and stem of Cleome viscose L., was seen to have high nematicidal activity
against M. incognita (Williams et al., 2003).
20
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The introduction of castor oil plant which is postulated to have naturally occurring
nematicides, reduced soil nematode population by 90 % while increasing the yield of
cassava and cocoyam by 29 % and 28 % respectively (Ugbaja 1997). The degree of
infection by nematodes on the tubers and corms were also reduced.
A number of plant species documented as Chinese traditional medicine have shown
nematicidal properties (Ferris and Zheng 1999). The plant parts tested included roots,
rhizomes and bulbs, stems, leaves, seeds, bark as well as whole plants. Some of the
compounds identified in many of these plant extracts include alkaloids, glucosides,
glycosides, organic acids and essential oils (Hsu et al, 1985; Huang 1993).
Application of molasses to soil by sprinkler irrigation system and by overhead boom
sprayer, lowered soil populations of reniform nematodes and caused a marked
improvement in plant growth and fruiting of papaya (Schenck 2001). Molasses supplys
carbohydrate therefore alters the carbon-nitrogen ratio, which affects the soil microbial
ecology causing a decrease in populations of plant parasitic nematodes, on the other hand
having favorable effects on plant growth. The efficacy of molasses for the control of root
knot nematodes was comparable to that of the chemical nematicide fenamiphos.
Soil amendments with raw and burnt rice husks in combination with solarization had
shown to decrease nematode populations in nurseries. Tea waste, coconut husks and by-
products from sugar cane when used as surface mulch or incorporated into soil managed
to decrease nematode populations in nurseries as well (UNDP, n.d.). Organic
21
Potential Application of Kava (Piper methysticum F.) in Nematode Control
amendments with pine bark, velvet bean and kudzu at the rate of 30 g/kg effectively
decreased nematode population (Chavarria-Cavajal et al., 2001).
Crop residues of maize, panicum and velvet bean were found to decrease the population
of plant parasitic nematodes as well (McSorley and Frederick 1999). Fresh neem leaf,
neem cake, tobacco waste, fresh plant material of wild sunflower, Adathoda vesica and
Vetiveria zizaniodes when used as mulches effectively reduced nematode populations
(Ngigi and Ndalut 2000).
1.7.3 Compounds Isolated from Plants with Nematicidal Activity.
A number compounds have been isolated from plants with antagonistic effects towards
plant parasitic nematodes. Phytochemicals with nematicidal and insecticidal properties
include polythienyls, isothiocyanates, glucosinolates, cyanogenic, glycosides,
polyacetylenes, alkaloids, lipids, terpenoids, sesquiterpenoids, diterpenoids, quassinoids,
steroids, triternoids, simple and complex phenolics and several other classes (Chitwood
1993, 2002).
Furfural (2-furfuraldehyde) (21) is a liquid found in many essential oils from plants and
in fruit juices, alcoholic beverages and bread. It is used in food industries as flavor
compositions. Insecticidal properties of furfural have already been established (Flor
1926; Raeder et al, 1925). Fungicidal properties of furfural were first reported in 1926
(Rodriguez - Kabana et al., 1993). Soil amendments with furfural have been known to
22
Potential Application of Kava (Piper methysticum F.) in Nematode Control
control southern blight in lentil. When furfural was added to soil at 0.1 - 1.0 ml/kg,
populations of Meloidogyne arenaria and Pratylenchus brachyurus were suppressed
(Rodriguez - Kabana et al., 1993). Fewer galls on the roots and an increase in yields
were noted.
Figure 1.6: Structure of 2-furfuraldehyde
Furostanol glycosides extracted from cell cultures of Dioscorea deltoidea decreased the
susceptibility of infection by root knot nemtodes (M. incognita) in tomato and cucumber
plants (Zinovieva et al., 1997). These plants when treated with furostanol glycoside
showed a five-fold decrease in overall nematode population. The female nematodes
present were smaller and there was a shift in the sex ratio towards more males.
Phytoecdy- steroid (20-hydroxyecdysone) (22), present in plants, caused fatalities to plant
parasitic nematodes on direct exposure. This phytoecdy- steroid is in fact the moulting
hormone of invertebrates and probably nematodes as well. Abnormal molting,
immobility, reduced invasion, impaired development as well as death were some of the
effects of this compound observed on plant parasitic nematodes (Soriano et al., 2002).
23
Potential Application of Kava (Piper methysticum F.) in Nematode Control
HO
Figure 1.7: Structure of 20-hydroxyecdysone
23
CHO
3 24
HC
CH 3 OH 25 HC
Figure 1.8: Structures of benzaldehyde, citral, furfural, menthol and α-terpineol
Other naturally occurring botanical compounds that have been tested for nematicidal
properties are benzaldehyde (23), citral (24), furfural (21), menthol (25) and α-terpineol
(26). These compounds are used commercially for perfume and flavor production. When
these compounds were mixed with soil at rates ranging from 0.1 to 0.5 mL/kg,
populations of M. incognita decreased in both the roots and the soil. The health of the
plants was not affected in any way by these compounds. Application of citral and
menthol caused significant increase in plant heights as well (Bauske et al., 1994).
24
Potential Application of Kava (Piper methysticum F.) in Nematode Control
1.8 Aims and Objectives of Present Study
This study focused to obtain freeze-dried polar extracts of Fiji and Vanuatu kava (Piper
methysticum), exhaustively remove ethanol solubles from this extract to attain highly
polar extract and to separate this extract into fractions using semi-prep HPLC.
Additionally, to culture RKN on tomato plants to obtain populations of juveniles and
eggs for bioassays, test the extracts for nematicidal activity in vitro,to conduct soil
amendment experiments with powdered root material of kava in pot experiments and to
compare the difference in bioactivity of Fiji and Vanuatu Kava.
25
Potential Application of Kava (Piper methysticum F.) in Nematode Control
CHAPTER 2
2.0 KAVA EXTRACTIONS AND SEPARATION
2.1 INTRODUCTION
The well known active ingredients in kava, the kava lactones are documented for their
antibacterial, antifungal and amoebicidal properties in addition to various effects on the
central nervous system of humans (Singh 1986; Sonfi et al., 1983; Sotheeswaran 1987;
Xuan et al., 20031; Walji 1998; Whitton et al., 2003). No reported work has been found
on the nematicidal properties of kava.
In this study various extraction and subsequent separation procedures conducted for the
preparation of samples for testing the nematicidal properties of kava are described. The
first extract of kava was the water extract, which would be deemed to contain water
solubles. This was one of the kava samples tested in this study and has been referred to as
Sample X. Sample X was further purified by removing the ethanol solubles in
preparation for the highly polar testing material, referred to as Sample Y. Semi-
preparative HPLC techniques, as described under section 2.2.4 were involved to further
fractionate sample Y into its subsequent fractions that is Samples Y1, Y2, Y3 and Y4.
The above samples were subjected to various bioassays for evaluating the nematicidal
properties of kava.
26
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.2 METHODOLOGY
2.2.1 General Methodology
Commercial samples of dried (powdered) kava root material from Fiji (FJ) and Vanuatu
(VA) cultivars were bought from Suva Market and from a local importer respectively.
The exact cultivar of these kava samples were not known but the FJ kava used in this
research was the one which is locally referred to as Kadavu kava while VA kava was
known as Vanuatu kava only in the local market.
Solvents used for extraction were of high purity at purchase and were redistilled before
use. Laboratory research grade water was used. Solvents and samples for use in HPLC
were membrane filtered (0.45 μm cellulose acetate filter) and ultrasonically degassed
prior to analysis. Samples were freeze dried (where necessary) using Dynavac
Engineering FD3 freeze drier and BUCHI Rotavapor R-114 equipped with BUCHI
Waterbath B-480 was used for rotor-evaporation.
Sections 2.2.2 to 2.2.4 outline the extraction procedure. (Refer to Appendix 1 for a flow
diagram summary).
27
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.2.2 Aqueous Extraction
About 500 g of powdered kava root material was soaked in approximately 1.5 L of water
and left at ambient temperature. After 24 hrs it was filtered using cheesecloth to obtain a
muddy colored suspension, which was first frozen followed by freeze-drying to get a
solid sample of mass of about 62 g. The residue (brown in color) was stored at 4 °C in the
refrigerator for use in bioassays. This extraction was carried out for both FJ and VA kava
and the samples obtained are referred to as sample X-FJ and X-VA hereafter. For FJ
kava, two samples X, i.e. X-FJ (stored) and X-FJ (fresh) were tested for juvenile
mortality abilities.
2.2.3 Removal of Ethanol Solubles
A 20 g sample of the freeze-dried residue, sample X was subjected to exhaustive soxhlet
extraction with 200 mL of absolute ethanol, for a period of 5 hrs. This was repeated four
to five times until the extraction solvent ran clear, that is the original color of the solvent
(ethanol). The remaining residue (totally devoid of ethanol solubles) was air dried in a
fume cupboard to get rid of any traces of ethanol from it. This residue (for both FJ and
VA kava) has been referred to as sample Y-FJ and Y-VA, and was stored at 4 °C in the
refrigerator for use in various bioassays and for semi-prep HPLC fractionation. Sample
Y-FJ was obtained from sample X-FJ (fresh).
28
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles
The final ethanol extract from soxhlet was dried in vacuo and redissolved in a known
volume of dichloromethane (DCM) to make a standard of approximately 1880 ppm. This
sample was analysed by a Waters (c) 515 HPLC equipped with a 2487 Dual Wavelength
UV absorbance detector set at 254 nm. Econosil Silica 10μ column of size 250 mm x 4.6
mm (length and internal diameter), mobile phase composition of 70 % n- hexane and 30
% ethylacetate and attenuation of 256 was used for analysis.
2.2.4 Separation of Sample Y
Organic analysis of kava has been highly restricted to the isolation and characterization
of kava lactones. Very little research has been done on the aqueous extract or the water
soluble components of kava and definitely none in which attempts have been made to
fractionate and isolate water-soluble components. Hence there was no available
information on the HPLC parameters to be used to achieve separation of the compounds
in sample Y for fractionation and isolation. Therefore method development for
identifying the best chromatographic parameters was a major part of this section.
29
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.2.4.1 Identification of λ max
Sample Y was dissolved in distilled water and placed in a quartz cuvette. Using Perkin-
Elmer Lambda 16 UV- Vis Spectrometer, the UV- Vis spectrum of this sample was
obtained in the range of 200 and 800 nm.
2.2.4.2 Mobile Phase Composition Determination for separation of Sample Y using
Thin Layer Chromatography
Various mobile phase compositions were tested to identify the composition, which gave
the best separation of the peaks. The three commonly used solvents (water, acetronitrile
and methanol) in reverse phase HPLC were tested for the best possible separation of the
compounds in Sample Y.
TLC of Sample Y was performed to identify the best condition for semi - preparative
HPLC fractionation. TLC of this sample was carried out using 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 plates.
The best separation in TLC analysis of Sample Y 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 tested that did show some
30
Potential Application of Kava (Piper methysticum F.) in Nematode Control
separation were various compositions of acetonitrile: methanol, methanol: water and
acetonitrile: water: methanol.
2.2.4.3 Gradient HPLC Analysis of Sample Y
A Waters HPLC system comprising of 1525 binary HPLC pump, 717 plus autosampler,
2996 photodiode array detector aided with Empower software was used for gradient
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 were used as the mobile phase.
Sample Y was dissolved in the appropriate mobile phase and in water for injection.
Analysis was carried out at different flow rates along various gradients 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.
The best separation was achieved at a flow rate of 0.5 mL/min. UV detection between the
range from 200 - 400nm gave the best detection at 210 nm. The use of acetonitrile:
methanol (80: 20 and 90: 10) as mobile phase gave good separation but column blocking
resulting in build up of high pump pressure was noted. This solvent composition was
considered not appropriate for analysis since column damage and pump damage could
have occurred.
31
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The solvent system of acetonitrile: water (90: 10, 85: 15, 80: 20 and 75: 25) produced
similar problem. A three solvent system of acetonitrile: water: methanol in various ratios
was used next. Some separation was achieved initially but high pump pressure was
observed afterwards. Therefore, this solvent system was not considered suitable for
analysis as well. Since sample Y is mainly composed of highly polar components,
complete elution was a problem (column blockage and excess build up of pump
pressure). Prior purification using column chromatography could have eliminated this
problem however, it was not engaged in this study. A highly polar solvent system was
thus regarded necessary for good separation.
The water composition in the mobile phase with either acetonitrile or methanol was thus
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 210 nm gave a relatively better separation without much
pressure problems. These parameters were then used for semi-prep analysis.
2.2.4.4. HPLC Separation of Potential Nematicidal Compounds from Sample Y
The suitably identified HPLC analytical parameters (mobile phase; 1: 3, acetonitrile:
water, flow rate of 0.5mL/min, UV detection at 210nm and an attenuation of 256) were
used for fractionation of sample Y using semi- prep HPLC. Chromatogram obtained
using these parameters is shown in Figure 2.3. The first fraction was collected between
12.3 to 17 mins, second from 17 to 21 mins, third between 21 to 23 mins and fourth,
32
Potential Application of Kava (Piper methysticum F.) in Nematode Control
which was a single, slightly broad peak around 31 mins. The volume of sample injected
was 200 μL and concentration was 0.01 g/mL.
These fractions were collected in vials and transferred in bottles which were kept at 4 °C
in the refrigerator. Once the bottles were full, the solutions were transferred to 500 mL
plastic beakers and frozen before freeze-drying. The dry residue obtained were weighed
and transferred to vials. The four fractions collected from samples Y-FJ and Y-VA were
labeled as Y1-FJ, Y1-VA,Y2-FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA and kept in
the refrigerator at 4 ºC for use in bioassays and for NMR analysis.
2.2.5 H-NMR Analysis of Fractions of Sample Y
analysis was conducted on the four fractions obtained. The H-NMR was
conducted at University of New England. Bruker Avance 300 Spectrometer and a 5 mm
inverse H/BB probe with z-gradient were used. Spectra were run in D2O at 30 °C and
128 scans were collected for the individual fractions.
33
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.3 RESULTS AND DISCUSSION
2.3.1 HPLC analysis of Sample Y to verify the removal of Ethanol Solubles
The final ethanol extract obtained from soxhlet was analyzed using normal phase HPLC
for the presence of any ethanol solubles. The peaks on the chromatogram were used as a
guide to verify that the ethanol solubles have been removed from sample X. The
following figures (2.1 and 2.2) show the chromatograms used for comparison.
:=; / \^.--'
I
I j
/ i
IT! I
/ \
Figure 2.1: HPLC chromatogram showing peaks of the ethanol solubles
Figure 2.2: HPLC chromatogram of the final ethanol extract from soxhlet
34
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The compounds which were detected in the first ethanol extract were not detected in the
final ethanol extract, as can be observed by the absence of the peaks in figure 2.2. It can
be stipulated that exhaustive soxhlet extraction removed ethanol solubles, leaving behind
the highly polar compounds that would be present in sample Y.
2.3.2 H-NMR Analysis of Fractions of Samples Yl, Y2, Y3 and Y4
The chromatogram in figure 2.3 shows the peaks which were used as a guideline to
isolate fractions Y1, Y2, Y3 and Y1, Y2, Y3 and Y4 from sample Y.
Figure 2.3: HPLC chromatogram for sample Y showing the groups of peaks which
were isolated
The four fractions were subjected to H-NMR analysis. The H-NMR spectra (Appendix
1) of these fractions as expected, were not very conclusive towards identification of the
structure of the compounds but some functionalities could be deduced. The presence of
anomeric protons was indicated by the peaks at 4.94 ppm and 5.34 ppm in fractions Y1
35
Potential Application of Kava (Piper methysticum F.) in Nematode Control
and Y2, and at 4.94 ppm and 5.1 ppm fractions Y3 and Y4. The absorbance around 3.0
ppm - 4.0 ppm in all the fractions indicates resonance resulting from the presence of
sugars around. The singlets and triplet around 1.1 ppm-1.3 ppm in all the fractions
indicate the presence of methyl group. The large peak at 4.66 ppm is most likely that of
water resulting from the exchange of the sugar protons with deuterium (D2O), as well as
residual H20 in D2O and possibly in the sample. Peak at 5.41 ppm could be that of DCM,
which was used to clean the tubes. Structure of the compounds present in the four
fractions could not be identified since individual compounds were not isolated. Presence
of large water and DCM peak also made structural elucidation difficult.
These results when taken together with the fact that fractions were highly polar, presence
of glucosidic compounds in addition to carbohydrates can be suggested in fractions Y1,
Y2, Y3 and Y4. Many plants produce glycosylated compounds as secondary metabolites.
Glycosylated compounds are known to be better stored within plant vacuoles and are less
reactive toward other cellular components (Pridham 1965).
36
Potential Application of Kava (Piper methysticum F.) in Nematode Control
2.4 CONCLUSION
The procedure outlined in this chapter made possible the separation of various extracts of
kava which were evaluated for nematicidal activity as will be reported in chapter 3.
Sample Y was isolated into sub-fractions Y1, Y2, Y3 and Y4 using RP semi-prep HPLC
however individual compounds could not be isolated using the procedure identified in
this research. Since the sample was not purified prior to subjecting to fractionation, build
up of column pressure was noted making it impossible to further isolate the fractions.
The H-NMR data of the fractions suggests the probability of the presence of glucosides
as postulated by Naiker and Prasad (2005) and Naiker et al. (2006) in addition to
carbohydrates (Sotheeswaran et al., 1998). Actual structures of the compounds could not
be deduced due to the complexity of the fractions. Each fraction consists of components
of slightly different polarities. This was seen by the clustering of groups of peaks in the
chromatogram.
The following chapter reports the bioactivity tests of various extracts of kava obtained.
37
Potential Application of Kava (Piper methysticum F.) in Nematode Control
CHAPTER 3
3.0 BIO ASSAYS
3.1 INTRODUCTION
The activity of kava and its subsequent extracts and fractions on nematodes can be
demonstrated through bioassays. Testing of the isolated fractions will narrow down the
chemistry of nematicidal compounds found in kava. Nematicidal activities of chemicals
are commonly evaluated through in vitro testing, where nematodes are in direct contact
with test chemicals or through amendments in soil. In soil amendment experiments
chemicals are mixed in soil in which susceptible plants are grown and inoculated with
nematodes.
The effect of different extracts of kava obtained (as described in chapter 2) was tested in
vitro for mortality of juveniles and suppression of egg hatching of root knot nematodes.
Samples X and Y for both FJ and VA kava were tested on juveniles as well as eggs at
different concentrations while samples Y1, Y2, Y3 and Y4 were only tested on juveniles
at one concentration due to the small amount of samples obtained. Sample X is the
aqueous extract, sample Y is the highly polar extract i.e. sample X less ethanol solubles
and samples Y1, Y2, Y3 and Y4 are fractions isolated from sample Y using semi-prep
HPLC.
38
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Furthermore, dried kava root powder of Fiji and Vanuatu kava was used as the testing
material for the soil amendment experiments. Kava powder at different concentrations
and after degrading for various periods were used in pots for these experiments.
Subsequently, tomato seedlings were planted and inoculated with juvenile nematodes in
these experimental pots. The efficacy of FJ and VA kava varieties towards the control of
root knot nematodes i.e. its nematicidal activity and their phytotoxic effect (any
detrimental effect) on the tomato plants was evaluated by observing the degree of root
infection.
39
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.2 METHODOLOGY
3.2.1 General Methodology
Nematode cultures had been set up for use in the various bioassays in this research. Visits
were made to vegetable farms and kitchen gardens along the Suva - Nausori corridor to
identify plants infected with root knot nematodes (RKN). Infected plants were identified
by randomly pulling out plants which looked sickly and looking for knots similar to those
in Figure 3.1. An infected farm at Nailuva road was identified and samples for raising
cultures were obtained from there.
'
Figure 3.1: Tomato roots infected by RKN as viewed with naked eyes
40
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.2.2 Nematode Culturing and Species Identification
Infected roots collected were washed in distilled water in a beaker, cut in pieces and
transferred to a glass Petri dish with clean distilled water. The knots were observed under
a stereoscopic microscope in the Petri dish to identify protruding egg sacs (masses)
attached to females. Each egg sac was transferred to separate cavity blocks and the
respective female was transferred to 0.9 % KCl (saline) solution before transferring to
lactic acid solution. The saline solution maintains equilibrium of the fluid inside and
outside the body of the female nematode and the lactic acid helps in clearing the body
fluid so that it is easier to identify the perineal pattern on the posterior portion.
Female nematodes were cut in half and the anterior portion was discarded. The posterior
portion was further trimmed around the perineal parts to obtain a small piece of the body
tissue with the perineal pattern. The perineal portion was transferred on to a drop of
glycerine on a slide and covered with a cover slip. The slide was sealed and left to dry
before labeling and observing under compound microscope to identify the species of the
root knot nematode. Perineal portions are like fingerprints which are different for
different nematode species. Observation of the adult (females) and juvenile stage
revealed that the nematodes infecting the plants were from Meloidogyne genus. Based on
the observation of the morphology of perineal patterns (Taylor and Sasser, 1978) the
nematodes infecting the plants were tentatively identified to be from the Meloidogyne
incognita group.
41
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The egg sacs attached to females identified tentatively as M. incognita were transferred to
clean distilled water in a 3.5 inches, disposable laboratory Petri dish and left in a BOD
(biological oxygen demand) incubator at 28 °C until the juveniles had hatched.
Suspensions of hatched juveniles were concentrated by allowing it to settle for few hours
in a beaker before excess water was removed from the top gently, using a Pasteur pipette,
being careful not to disturb the sediment at the bottom of the beaker.
Tomato seedlings of small fry variety were inoculated by planting them in sterilized soil
and sand mixture (5:1 ratio) to which this concentrated juvenile suspension had been
added. The tomato plants used were three weeks old and had been raised in sterilized soil.
These plants were allowed to grow undisturbed for about two months in the plot land
with frequent watering to allow the nematode population to establish. Juvenile population
and egg masses obtained from these culture plants were used for bioassays described in
the research.
3.2.3 Sample Preparation
The samples to be tested for nematicidal properties were dissolved in water at different
w/v (g/mL) concentrations. FJ kava had two X samples that is X-FJ (stored) and X-FJ
(fresh). Sample X-FJ (stored) had been stored in the refrigerator at 4 °C for six months
while sample X-FJ (fresh) had been freshly extracted.
42
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Samples X and Y of both FJ and VA kava were tested at 1 % and 2 % concentrations
while in some cases as indicated in the results, 0.5 % concentration was also tested. The
activities of 2 % concentration of these samples were compared with 0.5 % concentration
of gallic acid (standard). The fractions of Y samples i.e. Y1, Y2, Y3 and Y4 of FJ and
VA kava were tested at 0.1 % concentration and compared with gallic acid at 0.1 % w/v
concentration.
Samples were added to distilled water in conical flasks and shaken for 20 minutes using
Stuart Scientific Flask Shaker SF 1 to assist in dissolving the samples faster.
3.2.4 Laboratory Assays of Kava Extracts
The in vitro experiments were conducted for juvenile mortality and suppression of egg
hatching. The second stage juvenile (J2) populations and the egg masses needed for these
experiments were obtained from the maintained Meloidogyne sp. cultures. The
experiments for samples X and Y were performed in 3.5 inches disposable laboratory
Petri dishes, in which grids of 5 x 5 mm had been made using a needle while for samples
Y1, Y2, Y3 and Y4 were performed in 2 inches disposable Petri dishes with grids, 2.5 x
2.5 mm. The grids were marked to aid in the counting process.
43
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.2.4.1 Juvenile Mortality Tests
Activity of samples X-FJ (stored), X-FJ (fresh), X-VA, Y-FJ, Y-VA, Y1-FJ, Y1-VA, Y2-
FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA was evaluated on second stage juveniles
(J2). All the bioassays were conducted at 28 ºC in BOD incubator while X-FJ (stored) and
X-FJ (fresh) were also tested at room temperature. Juveniles were obtained by allowing
the egg masses to hatch in distilled water in a BOD incubator for 7 to 10 days. J2
suspension was standardized to 100 juveniles/mL by either concentrating or diluting the
suspension.
Experiments for samples X and Y of FJ and VA were set using 10 mL of the respective
concentrations (2 %, 1 % and 0.5 %) of the test solutions and 1 mL juvenile suspension
per Petri dish. For samples Y1, Y2, Y3 and Y4, 3 mL of 0.1 % test solution and 1 mL of
juvenile suspension was used. The 0 % concentration (distilled water) served as the
control for comparison.
Quantitative observation of the treated juveniles was made after 24 hrs and 48 hrs of
treatment after which the juveniles were transferred to distilled water. This was done by
transferring the contents of the Petri dish (juveniles and test solutions) in a beaker and
diluting it with distilled water. The beakers were left for two to three hours before
removing excess water using a Pasteur pipette. This was repeated three to four times to
further dilute the samples. The remaining mixture was transferred back to the respective
Petri dishes and left in the incubator for another 24 hrs before re-observation under the
44
Potential Application of Kava (Piper methysticum F.) in Nematode Control
microscope. This was to verify whether the effect of the samples tested was permanent or
not. All tests were conducted in seven replicates.
The percentage mortality of the juveniles was determined by counting the number of
dead and live juveniles at each observation using manual tally counters. As the Petri
dishes were scanned from one grid to another, the tally counters, one for dead and one for
live juveniles was clicked when a juvenile was spotted. Mortality of the juveniles was
indicated by immobilization and dead (slightly curved to almost straight) posture of the
juveniles. The results obtained after revival in distilled water was subjected to analysis of
variance using regression with SPSS version 12.0.1 for windows software.
Figure 3.2: Live juveniles in motion in water as viewed under 40 x magnification
45
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Figure 3.3: Dead juveniles viewed under 40 x magnification (note the slightly curved
to almost straight posture)
3.2.4.2 Egg Hatching Experiments
Egg hatching experiments were conducted for samples X-FJ (fresh), X-VA, Y-FJ and Y-
VA at 1 % and 2 % concentrations in seven replicates. The activity of 2 % concentration
was compared with 0.5 % concentration of gallic acid. Experiments were set using 15
mL of the test solutions and two approximately equal sized egg masses per Petri dish at
28 °C in BOD incubator. These Petri dishes were replenished with about 8 mL of test
solutions on the third and sixth day to prevent them from drying.
46
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Quantitative observation under the microscope was made on the third, sixth and eighth
day for sample X-FJ (fresh), since majority of the eggs hatch within 7 days. Since the
results of the third sixth and eight day did not differ much, observations for the other
experiments were recorded on the third and seventh day only. Total number of hatched
juveniles as well as the number of dead and live juveniles was counted with a manual
tally counter and recorded. Results obtained were analyzed between subjects for
multivariate analysis using general linear model with SPSS software.
3.2.4.3 Comparison with the standard (Gallic Acid)
The activity of the kava extracts towards juvenile mortality and inhibition of egg hatching
was compared with the standard, gallic acid. These were separate experiments conducted
at 2 % concentration of the test solutions with 0.5 % concentration of standard. Gallic
acid was chosen as the standard since it is one of the phenolics present in methanolic
extracts of kava.
For comparing the activity of samples Y, a more branched or maybe a glycosidically
bound polyphenol should have been used since such compounds have been postulated to
be present in sample Y (Naiker and Prasad 2005). This is because extraction with ethanol
in preparation of sample Y would remove low molecular weight compounds, chemically
similar to gallic acid since these would be soluble in ethanol.
47
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.2.5 Pot Experiments (Soil Amendment Experiments)
The soil amendment experiments were conducted to evaluate the effect of kava on
nematodes growing on plants and its effect on plants. These experiments were carried out
using dried kava root material as soil amendment before planting and inoculating tomato
seedlings with juvenile nematodes.
3.2.5.1 Preparation of Pots and Soil for Experiments
The soil amendment experiments were carried out in 9 x 9 cm (diameter and height)
plastic pots, which were washed and dried prior to use. The soil to be used for the
experiment was mixed with sand in a 5: 1 (soil and sand) ratio and sterilized in an
autoclave at 80 °C and 20 psi for 1 hr to terminate all the pests in the soil including any
nematodes that may have been present. The sand and soil mixture used per pot was 400 g.
3.2.5.2 Experimental Setup
Powdered kava root material was used for amendment of soil in these experiments. Four
different experiments (where kava was degraded in the soil for different time periods)
were set up for each kava sample. Each of these experiments had four different
treatments and seven replicates.
48
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The different treatments were different kava concentrations in the soil which were 5 %, 2
%, 1 % and 0 % (control) weight to weight (w/w). These concentrations were obtained by
manually mixing 20, 8, and 4 g of kava powder respectively to 400 g soil for each pot and
only sterilized soil as control. A few (8 to 10) grains of urea, a commercial fertilizer was
also added to the soil on the day of mixing kava and every 4 weeks thereafter. The pots
with the different kava concentrations were placed randomly on the benches in the plot
land.
The four different experiments were for 0, 2, 4, and 6 weeks of degradation of kava in
soil. This was achieved by planting seedlings on the day of adding kava powder to soil,
and 2, 4, and 6 weeks after adding kava to soil respectively. The pots were watered
frequently to assist in the degradation process.
3.2.5.3 Planting of Seedlings and Inoculating
Tomato seedlings were raised in sterilized soil at atmospheric temperature and pressure.
Experiments were set using three weeks old seedlings of small fry variety of tomato that
had been raised in sterilized soil. After the respective periods of degradation, a pit was
made in each pot and 2 mL of standardized (100 juveniles/mL) J2 suspension was added.
One seedling per pot was planted and allowed to grow without much interference except
for watering every second day. These experiments were terminated after 52 days as this
gives sufficient time for RKN to complete at least one life cycle and gall formation by the
next generation .
49
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Figure 3.4: Kava powder mixed in soil and left for degrading in the plot land
50
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Figure 3.5: The author planting the tomato plants in the pots after inoculating with
juvenile nematodes
3.2.5.4 Plant Observations
On the 52nd day the plants were carefully removed from the pots avoiding breaking off
of any roots. These were then washed in water gently, preventing washing away of the
egg masses and were left on blotting paper for a few minutes before observing the degree
of nematode infection. Using magnifying lenses the number of galls were observed and
recorded. The plant heights and root lengths were also recorded. The results obtained
were compared to see the effect of different kava concentrations and the different
degradation periods on root galling by the nematodes on the plants.
51
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3 RESULTS AND DISCUSSION
3.3.1 Laboratory Assays of Kava Extracts
3.3.1.1 Juvenile Mortality Experiments
Juvenile mortality experiments were carried out for samples X-FJ (stored), X-FJ (fresh),
X-VA, Y-FJ, Y-VA, Y1-FJ, Y1-VA, Y2-FJ, Y2-VA, Y3-FJ, Y3-VA, Y4-FJ and Y4-VA.
Samples X-FJ (stored), X-FJ (fresh) and Y-FJ were tested at room temperature as well as
at controlled temperature (28 °C).
a) FJ Kava Extracts Tested at Room Temperature
Sample X (stored), which was stored in the refrigerator for about six months, was
effective within 24 hrs of treatment time at both 2 and 1 % concentrations (Table 3.1).
Results summarized in Table 3.1, shows 100 % mortality at the 2 % concentration and 34
% mortality at 1 % concentration. Percentage mortality at both concentrations remained
almost same even after 48 hrs of treatment. Observation after 24 hrs of revival in
distilled water illustrated 30 % (at 2 % concentration) and 14 % (at 1 % concentration)
revival of the juveniles. That is, % mortality after revival decreased to 70 and 21 % in 2
and 1 % treatments respectively. This indicates that the effect of sample X (stored)
reversible to some degree even after 48 hrs of treatment. Analysis of variance gave P <
52
Potential Application of Kava (Piper methysticum F.) in Nematode Control
0.001 for both time and concentration effects thus indicating the significant effect of
these on observed mortality.
Sample X (fresh) produced juvenile mortality after 24 hrs of treatment at both
concentrations. Observed mortality increased after 48 hrs of treatment for the two
concentrations. Percentage mortality was lower than sample X (stored) after 24 and 48
hrs of treatments. After 24 hrs of exposure to distilled water, juvenile mortality at 2 %
concentration increased from that observed after 48 hrs of treatment, while it remained
same for the 1 % concentration. This indicates that the toxic effect had been initiated after
48 hrs of treatment and was permanent since transferring the juveniles to distilled water
did not help them to recover. The observed was P < 0.001 indicating a significant effect
of concentration and treatment time on juvenile mortality.
The final mortality (after revival in distilled water) caused by sample X (fresh) was
higher than sample X (stored). The 2 % concentration showed more significant results
than the 1 % concentration. The variation in the efficacy of samples X (stored) and X
(fresh) could be due to some chemical changes which may have occurred over the storage
period. However, this can not be concluded with certainty since these were single trials
only.
Sample Y was tested at 2 %, 1 % as well as 0.5 % concentrations. Juvenile mortality was
noted in all tested concentrations which increased with increasing treatment time.
Treatment time is one of the important factors contributing towards the efficacy of
53
Potential Application of Kava (Piper methysticum F.) in Nematode Control
extracts since higher mortality was noted with longer periods of exposure to the
chemicals. When the juveniles were transferred to distilled water for revival, a significant
rise in mortality was observed for the 2 % test solution. This indicates that the activity of
sample Y (especially at 2 % concentration), on the juveniles was slow but permanent.
Relatively high mortality rates were observed for the control as well. The temperature
(room temperature) at which these treatments were kept was not constant, thus it could
possibly be contributing towards the variations in the results.
Table 3.1: FJ kava extracts tested for juvenile mortality at room temperature
Treatment
Time
24hrs
48hrs
24 hrs revivalin distilledwater
Sample X-FJ (stored)
2%
100
100
70
1%
34
35
21
0.5*%
X
X
X
Control
3
5
5
Sample X-FJ (fresh)
2%
67
78
83
1%
26
29
29
0.5*%
X
X
X
Control
3
4
6
Sample Y
2%
20
27
99
1%
14
22
21
0.5%
21
30
28
Control
11
10
11
* - this concentration was not tested.
Table 3.2: Analysis of Variance Results for FJ Kava Samples tested at Room
Temperature
Sample Tested
X-FJ (stored) at roomtemperature
X-FJ (fresh) at room temperature
Degrees ofFreedom
2, 15
2,15
Observed Fvalue
16.2
15.2
Observed Pvalue
0.0000
0.0000
54
Potential Application of Kava (Piper methysticum F.) in Nematode Control
b) FJ Kava Extracts Tested at 28 °C
All three samples of FJ kava (X (stored), X (fresh) and Y) were tested for juvenile
mortality under controlled temperature. This was done to eliminate the effect of
fluctuating temperature, if any on observed mortality. Effect of sample X (stored) on
juveniles was almost similar to that observed at room temperature. Concentration of test
solution as well as treatment times significantly affected juvenile mortality since P <
0.0001. Mortality increased when transferred to distilled water, which was more
significant for the 2 % concentration. This indicated that the effect of the extract on the
juvenile was permanent and could not be reversed by isolating the juveniles from the
contact of chemicals.
Sample X (fresh) was tested at 2 %, 1 % and 0.5 % concentrations. The 1 % and 0.5 %
solutions presented very low mortality after 24 and 48 hrs of treatment but 2%
concentration was quite significant. When subjected to revival in distilled water, an
increase in mortality was noted for 2 % test solution, whilst 1 % and 0.5 % did not show
much variation. Percentage mortality observed after revival for both samples X (stored)
and X (fresh) was comparable for 2 % concentration. This indicates that storing the
sample at 4 °C did not cause any major chemical changes.
55
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The difference in activity observed in the experiments conducted at room temperature
(3.3.1.1), could have been more due to fluctuating atmospheric temperature than of any
chemical changes over the storage period. Nematodes are quite sensitive to temperature
and an optimum temperature between 25 to 32 °C is vital for their survival (Koenning et
al., 2004). Temperatures higher than 35 °C and lower than 20 °C usually lead to
mortality. Hence solarising, where soil temperatures are increased above 40 °C, has been
adapted as one of the mechanisms for nematode control. Statistical analysis gave P <
0.001, indicating significance of concentration and treatment times on mortality.
Sample Y showed low mortality at all concentrations at 24 hrs but values increased after
48 hrs. This indicates that sample Y had a slower effect on juveniles compared to sample
X. Significant effect of concentration and treatment times were noted on observed
mortality since P < 0.001. Some recovery of juveniles was noted when transferred to
distilled water. Therefore the ethanol solubles present in sample X that were removed for
the preparation of sample Y, can be said to be having a stronger and more permanent
effect on the juveniles than the compounds remaining in sample Y. Furthermore,
mortality was not only due to ethanol solubles but also the non-solubles or highly polar
compounds present in kava, since juvenile mortality was observed in treatments with
sample Y.
56
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Table 3.3: FJ kava extracts tested for juvenile mortality at 28°C
Treatment
Time
24hrs
48hrs
24hrsrevival indistilledwater
Sample X-FJ (stored)
2%
64
64
81
1%
6
12
16
0.5*%
X
X
X
Control
3
3
3
Sample X-FJ (fresh)
2%
70
70
82
1%
4
7
8
0.5%
3
5
5
Control
1
1
1
Sample Y
2%
13
75
74
1%
2
14
4
0.5%
1
4
1
Control
1
2
2
* Concentration not tested
Table 3.4: Analysis of Variance Results for FJ Kava Samples tested at 28 °C
Sample Tested
X-FJ (stored) at 28°C
X-FJ (fresh) at 28°C
Degrees ofFreedom
2,15
3,20
Observed F value
11.2
13.4
Observed P value
0.0001
0.0000
c) VA Kava Extracts Tested at 28 °C
Two samples of VA kava (X-VA and Y-VA) were tested. Sample X was effective at both
2 % and 1 % concentrations. Significance of concentration and treatment times were
obvious since P < 0.0001. Observations after 48 hrs of treatment presented higher
mortality for the higher concentrations. Mortality increased in both concentrations with
increasing treatment times (Table 3.5). When transferred to distilled water after 48 hrs of
57
Potential Application of Kava (Piper methysticum F.) in Nematode Control
exposure, juvenile recovery was noted in both concentrations. This indicated that the
effect of sample X could be reversed to some extent. The mortality observed may be due
to some other chemicals than that found in FJ kava since results for the two kava extracts
were not comparable. On the other hand, it could also be possible that more than one
compound is responsible for this activity which may be present in different
concentrations in FJ and VA kava.
Sample Y was tested at 2 %, 1 % as well as 0.5 % concentrations. All of the three
concentrations tested did not show much activity after 24 hrs of treatment but increased
slightly after 48 hrs. The observed P < 0.0001, indicating the significance of
concentration and treatment times. When transferred to distilled water for revival,
mortality rate increased in 2 % concentration while it decreased in 1 % and 0.5 %
concentrations. This indicated that 2 % concentration was the most effective
concentration for sample Y of VA kava. The 1 % concentration of sample X showed a
higher effect compared to the 1 % concentration of sample Y. Slightly higher mortality
was observed at the 2 % concentration of sample X than sample Y. Therefore it could be
said that ethanol solubles which were not present in sample Y had significant effect
towards mortality.
58
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Table 3.5: VA kava extracts tested for juvenile mortality at 28°C
Treatment Time
24 hrs
48 hrs
24 hrs revival indistilled water
Sample X-VA
2 %
68
92
70
1 %
23
69
37
0.5* %
X
X
X
Control
2
2
2
Sample Y-VA
2 %
4
12
62
1 %
3
8
4
0.5%
2
8
4
Control
1
2
2
* - this concentration was not tested
Table 3.6: Analysis of Variance Results for VA Kava Samples tested at 28 °C
Sample Tested
X-VA at 28°C
Y-VA at 28°C
Degrees ofFreedom
2,15
3,20
Observed F value
19.6
107.1
Observed P value
0.0000
0.0000
d) Comparison of sample X of FJ and VA Kava with the standard
The minimum concentration at which Gallic acid showed effective mortality was found
to be 0.5 % (w/v). This concentration was as effective as those for the 2 % concentrations
of sample X-FJ and X-VA on the juveniles. Mortality observed after 24 hrs of treatment
was higher for sample X-VA compared to that of sample X-FJ and the standard (Table
3.7). An increase in mortality was noted in all treatments after 48 hrs. When exposed to
distilled water for 24 hrs, mortality increased for X-FJ and gallic acid while for sample
X-VA some revival was noted. The mortality noted for the standard was in the range for
59
Potential Application of Kava (Piper methysticum F.) in Nematode Control
that of FJ and VA kava. Results obtained as mentioned above were similar to those for
the two kava extracts conducted separately (3.2.1.2 and 3.2.1.3).
Table 3.7: Comparison of the samples X of FJ and VA kava with gallic acid
Treatment Time
24 hrs
48 hrs
24 hrs revival indistilled water
2 % Sample X-FJ(fresh)
54
65
95
2 % Sample X-VA
76
81
80
0.5 % Gallic Acid
58
80
84
Control
2
2
2
e) Comparison of sample Y of FJ and VA Kava with the Standard
The 2 % concentrations of samples Y of FJ and VA kava were compared with 0.5 %
Gallic acid. Juvenile mortality after 24 hrs of treatment was similar for FJ kava extract
and standard while VA kava extract recorded lower mortality (Table 3.8). Subjecting
treated juveniles to distilled water showed some recovery of juveniles in the FJ kava
extract as noted in earlier experiment while mortality increased in VA kava extract and
the standard. The results in this experiment were similar to those observed for previous
experiments on sample Y (3.2.1.2 and 3.2.1.3).
Mortality results of samples X and Y are comparable with the results obtained for Gallic
acid. Gallic acid together with other phenolics has been reported in 80 % methanol
extract of kava and was observed to have allelopathic properties (Xuan et al., 20032).
60
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Table 3.8: Comparison of samples Y of FJ and VA kava with gallic acid
Treatment Time
24hrs
48hrs
24 hrs revival indistilled water
2 % Sample Y-FJ
75
86
83
2 % Sample Y-VA
50
51
65
0.5% Gallic Acid
76
87
89
Control
1
2
2
3.3.1.2 Juvenile Mortality Experiments of the Fractions
The four fractions isolated from sample Y-FJ were labeled as Y1-FJ, Y2-FJ, Y3-FJ and
Y4-FJ, while those isolated from Y-VA were labeled Y1-VA, Y2-VA, Y3-VA and Y4-
VA, in the order in which they eluted from the column. The column used had a non-polar
stationary phase; therefore the fractions which eluted later were less polar compared to
the earlier eluting ones. The activity of these fractions on juveniles were tested in vitro
experiments and compared with control and the standard. The fractions and gallic acid
(standard) were tested at 0.1 % (w/v) concentration.
Juvenile mortality increased with increasing times in all treatments. Statistical analysis of
both FJ and VA kava fractions gave P < 0.0001. Observations after 24 hrs of revival in
distilled water showed reversible effect of the two highly polar fractions (Y1 and Y2) for
both FJ and VA kava while the less polar fractions (Y3 and Y4) showed continued
mortality that is increase in mortality from that observed after 48 hrs of treatment. Gallic
acid showed similar activity to fractions Y3 and Y4 and was consistent with the effect
observed in earlier experiments.
61
Potential Application of Kava (Piper methysticum F.) in Nematode Control
The general trend in the activity of the four fractions isolated from FJ and VA kava
samples showed similar activity thus indicating that compounds with similar chemistry
were present in both FJ and VA kava. However, overall mortality was higher in sample
Y4-VA than Y4-FJ but was comparable for samples Y1-Y3 for the two kava samples.
Table 3.9: Analysis of Variance Results for FJ and VA Kava fractions tested at
28 °C
Sample Tested
Y1-FJ, Y2-FJ,Y3-FJ, Y4-FJ at28°CY1-VA, Y2-VA,Y3-VA, Y4-VA at28°C
Degrees ofFreedom
1,5
1,5
Observed F value
24.5
12.1
Observed P value
0.0000
0.0000
62
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Observed juvenile mortality over the treatmentperiod for FJ Kava Fractions
30
25
i" 205 15
0 .m-,Y1 Y2 Y3 Y4 Gallic acid Control
• 24 hr s 48 hr s 24 hrs of revival in distilled water
Figure 3.6: Juvenile Mortality for FJ kava fractions
Observed juvenile mortality over the treatment35 n period for VA Kava Fractions
30
25
20
15
10
5
0 IlkY1 Y2 Y3 Y4 Gallic acid Control
• 24 hr s 48 hr s 24 hrs of revival in distilled water
Figure 3.7: Juvenile Mortality for VA kava fraction
63
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.1.3 Egg Hatch Experiments
The efficacy of samples X and Y at 2 % and 1 % concentrations for FJ and VA kava on
suppressing egg hatch was investigated in vitro at 28 °C. The total number of juveniles
hatched was counted, taking into account the number of dead and alive. Percentage
hatching was calculated by taking the number of hatched juveniles in control as 100 %
and percentage mortality was calculated based on total number hatched.
A problem encountered during these experiments was that the Petri dishes started drying
over the observation period. This was overcome by adding about 8 to 10 ml of distilled
water on the 3rd and 6th day for samples X and Y of both FJ and VA kava. The
experiments where comparison was being made with standard, 8 to 10 ml of test
solutions were added instead.
a) Activity of samples X and Y of FJ and VA kava
The results as seen in Figure 3.8 and 3.9 shows that both 2 % and 1 % concentrations of
sample X-FJ (fresh) and X-VA suppressed egg hatching. Results for the activity of
sample X-FJ (fresh) was recorded on third, sixth and eighth day while for sample X-VA,
it was recorded on the third and seventh day only. The total number of hatched juveniles
was found to be increasing in 2 %, 1 % as well as the control over the counting period,
but the 2 % had the lowest increase while control had the highest. At 1 % treatment with
sample X-FJ (fresh), the number of juveniles on the 8th day was almost half that of
64
Potential Application of Kava (Piper methysticum F.) in Nematode Control
control while in the 2 % concentration it was less than one fifth. Mortality of the
juveniles was also noted in both the 2 % and 1 % concentrations and was seen that almost
50 % of the hatched juveniles were dead by the 8th day of observation. While for X-VA,
on the 7th day, 2 % concentration showed only about 7 % egg hatching and 1 % showed
around 19 % compared to the control, where a total number of 206 juveniles had hatched.
Moreover, mortality of the hatched juveniles was also noted in the 2 % and 1 %
concentration.
Sample Y-FJ also showed that both the concentrations tested were effective in
suppressing egg hatch (Figure 3.10). There was an increase seen for the total number of
hatched juveniles in the two test concentrations as well as the control over the two
observation days but was very slight in the 2 % concentration. Percentage hatching was
around 3 % in the 2 % concentration and 16 % for the 1 %. Mortality was also noted at
the higher concentrations.
Similar observations were made for sample Y-VA as seen in Figure 3.11. Both the
concentrations tested were effective in suppressing egg hatching. Observations on the 7th
day showed that there was no increase in the number of juveniles in 2 % concentration
but some increase was seen in the 1 % concentration.
Activity of the sample X when compared with sample Y for both FJ and VA kava
showed similar effect on egg hatching. For both FJ and VA kava, it was noted that the
number of hatched juveniles increased slightly over the experimental period for sample
65
Potential Application of Kava (Piper methysticum F.) in Nematode Control
X, while for samples Y there was hardly any increase. Hence sample Y could be said to
be more effective in suppressing hatching of root knot nematode compared to samples X.
Table 3.10: Results for Sample X-FJ (fresh)
3 rd Day
6th Day
8th Day
2%
%Hatching
5
15
18
%Dead
8
32
43
No. ofJuvenilesHatched
4
30
41
1%
%Hatching
21
38
53
%Dead
1
7
20
No. ofJuvenilesHatched
18
75
125
Control
%Hatching
100
100
100
%Dead
0
2
3
No. ofJuvenilesHatched
88
199
235
Table 3.11: Results for Sample X-VA
3rd
Day
7th
Day
2%
%Hatching
3
8
%Dead
100
28
No. ofJuvenilesHatched
2
15
1%
%Hatching
7
19
%Dead
58
15
No. ofJuvenilesHatched
4
39
Control
%Hatching
100
100
%Dead
0
1
No. ofJuvenilesHatched
59
206
66
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Treatment of Egg Masses with Sample X-FJ (fresh)
ITsO)O)LJJo5
120100
80
6040
20
0
2% 1% Control
Thir d D a y Sixth Day —*— Eighth day
Figure 3.8: Treatment of egg masses with sample X-FJ (fresh)
Treatment of Egg Masses with Sample X-VA
3re
O)
120
100
80
60
40
20
02% 1% Control
Third Day • Seventh Day
Figure 3.9: Treatment of egg masses with sample X-VA
67
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Table 3.12: Results for Sample Y-FJ
3rd
Day
7th
Day
2%
%Hatching
6
3
%Dead
12
89
No. ofJuvenilesHatched
11
13
1%
%Hatching
25
16
%Dead
1
13
No. ofJuvenilesHatched
42
68
Control
%Hatching
100
100
%Dead
0
5
No. ofJuvenilesHatched
168
425
Table 3.13: Results for Sample Y-VA
3rd
Day
7th
Day
2%
%Hatching
23
6
%Dead
55
93
No. ofJuvenilesHatched
28
28
1%
%Hatching
29
9
%Dead
3
67
No. ofJuvenilesHatched
36
44
Control
%Hatching
100
100
%Dead
0
4
No. ofJuvenilesHatched
125
474
68
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Egg
Hat
chin
g120
100
80
60
40
20
0
Treatment of Egg Masses
2% 1
Third Day -
with Sample Y-FJ
% Control
y S e v e n t h Day
Figure 3.10: Treatment of egg masses with sample Y-FJ
tchi
n
*OlUJ
S?
120
100
80
60
40
20
0
Treatment of Egg Masses with Sample Y-VA
2% 1%
Third Day —•—Seventh Day
Control
Figure 3.11: Treatment of egg masses with sample Y-VA
69
Potential Application of Kava (Piper methysticum F.) in Nematode Control
b) Comparison of Activity of Samples X and Y of FJ and VA Kava with Standard
Gallic acid was used for comparison of the activity of the two samples. Two separate
experiments were conducted to compare the activity of the X and Y samples with gallic
acid. Samples X-FJ (fresh) and X-VA were compared with standard in one experiment
while samples Y-FJ and Y-VA were compared in another. Both, samples X-FJ (fresh)
and X-VA, showed almost nil egg hatching on the 3rd day, while gallic acid showed about
5 % egg hatching compared to the control which had a total number of 155 hatched
juveniles (Figure 3.12). The 7th day results were still quite low for the two test solutions.
The standard as well did not show any significant increase in the number of hatched
juveniles hence lowering the percentage hatching for the 7th day with respect to that of
the control in which the numbers had almost doubled.
The results for samples X-FJ (fresh) and X-VA were similar to the separate experiments
carried out thus showing reproducibility and competence of the kava extracts in
suppressing egg hatch.
Samples Y-FJ and Y-VA as well as the standard showed some egg hatching on the 3rd
day. The number of hatched juveniles were almost same for the samples Y-FJ and Y-VA
but was little higher for the standard as per figure 3.13. The number of hatched juveniles
on the 7th day remained the same in all the experiments except the control, which showed
a three-fold increase.
70
Potential Application of Kava (Piper methysticum F.) in Nematode Control
It was noticed that the experiments in which test solutions were used to replenish the
drying of solutions, the number of hatched juveniles did not increase from that recorded
on day 3. While the experiments where water was added, allowed a slight increase in the
number of juveniles after the 3rd day. Hence it could be concluded that addition of water
diluted the effect of the chemicals thereby allowing some more eggs to hatch.
The observed nematicidal activity of the X samples can be due to any of the water-
soluble components including suspended kava lactones which are consumed as kava
drink. The kava lactones have been reported to have anesthetic effect on animals
including humans (Cambie and Ash 1994; Cordell 1998; Davis and Brown 1999 and
Walji 1998). Since nematodes are very small organisms with simple body systems, the
kava lactones could have led to irreversible paralyses and hence death of the nematodes.
The effect could not be on the digestive system of the nematodes since the J2 stage is the
infective stage and the nematodes do not feed at this stage, instead they use the stored
energy (Ferraz and Brown 2002; Weischer and Brown 2000). These lactones are also
known to have antifungal, antibacterial and amoebicidal activities (Singh 1986;
Sotheeswaran 1987; Walji 1998; Xuan et al, 20031).
Other water soluble compounds reported in kava and which may be responsible for this
nematicidal activity are glutathione, pigment molecules, bornyl esters and phenolic
compounds (Basko 2002; O'hara et al, 1965; Singh 1986; Smith 1979, 1983;
Sotheeswaran et al., 1998; Whitton et al, 2003; Wu et al, 2002; Xuan et al, 20032).
These compounds could possibly be present in samples X, Y and the fractions of both FJ
71
Potential Application of Kava (Piper methysticum F.) in Nematode Control
and VA kava. The phenolic compounds have been reported to be allelopathic activities
(Xuan et al., 20031). The nematicidal activity therefore can not be accredited to any one
of the compound present in kava. It could be due to any of the compounds mentioned
above or could be the result of the combined effect of a number of compounds.
72
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Comparision of Activity of Samples X of FJ and VA Kava withGallic Acid for Egg Hatching
O)
U)
UJ
120
100
80
60
40
20
0
2% Sample X-FJ 2% Sample X-VA 0.5% Galic Acid(fresh)
Control
• Third Day • Seventh Day
Figure 3.12: Comparison of activity of samples X of FJ and VA kava with Gallic
acid for egg hatching
IUJ
120
100
80
60
40
20
0
Comparison of Activivty of Sample Y of FJ and VA Kava withGallic Acid for Egg Hatching
*+++*+++*+++
*+++*+++*+++*+++*+++*+++*+++
WWWT
II2% Sample Y-FJ 2% Sample Y-VA 0.5% Gallic Acid Control
• Third Day • Seventh Day
Figure 3.13: Comparison of activity of samples Y of FJ and VA kava with Gallic
Acid for egg hatching
73
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.2 Pot Experiments (Soil Amendment Experiments) for FJ and VA Kava
3.3.2.1 No Degradation Allowed (0 weeks)
The average number of root galls formed by RKN decreased as the concentration of kava
was increased in the soil. Number of root galls shown for the 5 % concentration was from
one plant only since plants in the other replicates had died. Some plant mortality was also
noticed in the other treatments. Mortality increased with increasing concentration of kava
in soil. This indicates that in addition to being effective in reducing root galls, kava also
has some detrimental effect on plants especially at 5 % concentration. The plant height
measurements showed a decreasing trend with increasing kava concentrations but the
root length values were similar to control plants (Table 3.14 and 3.15). The average
number of galls in the control was 119 and all the plants were healthy in these pots.
Results indicate that when seedlings are planted in the soil on the day of addition of kava,
plant health is affected leading to death. The effect was more prominent as the
concentrations increased. Results for VA kava are comparable to that FJ kava. The
average of total number of galls in the control pots of FJ kava was 119 while for VA kava
was 62. In both of these experiments it was noticed that though nematode population
decreased by adding kava, there was some phytotoxic effects on the plants. The
compounds present in kava responsible for these activities can not be determined at this
stage.
74
Potential Application of Kava (Piper methysticum F.) in Nematode Control
TO
cre(D
ve.
<
12
10
8
6
4 -
2r\U
0 Weeks Degradation FJ
\ ^ ^\ \
\^ B
1 2
% Kava in Soil
—•— % healthy plants (live plants) —•—Average
Kava
^ >
5
of total number of Galls
80- 70
60504030
?n10
£reE>.
eal
i
Figure 3.14: Zero weeks degradation FJ kava
TO
ve.
gal
25 i
20
10
5
r\U i
0 Weeks Degradation VA
•
1 2
% Kava in Soil
—•— % healthy plants (live plants) —•—Average
Kava
-
of total
—•
5
number of Galls
60
50
40
30
20
10
Pla
ntH
ealth
y
Figure 3.15: Zero weeks degradation VA kava
75
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.2.2 Two Weeks of Degradation
The effect of degrading kava in soil for two weeks before planting and inoculating the
seedlings was observed. All three treatments showed a decrease in root galls. The control
of FJ kava recorded 34 as the average root galls while VA kava had 55. As in previous
experiments nematode population was dependant on the concentration of kava in the soil.
Plant mortality was not noticed for treatments by FJ kava while treatments by VA kava
caused some mortality at the higher concentrations. An increase in plant height was
noticed in all the treatments compared to control for FJ kava, while root length decreased
slightly. Treatments for VA kava led to a decrease in plant height as well as root lengths
when compared to the control.
3.3.2.3 Four Weeks of Degradation
Four weeks of degradation of kava in the soil showed positive results towards control of
nematode population. Root galls made by nematodes were quite less in treatment plants
compared to control. The control for FJ kava showed an average of 97 root galls while
VA kava showed 66. As observed in other experiments, higher concentrations were more
effective compared to lower ones. In regards to mortality of plants for FJ kava, 1 %
treatment did not show any prominent effect but in 2 % and 5 % treatments, one and two
plants respectively had died. For VA kava plant mortality remained same for 2 %
concentration but was also noted at 1 % concentration. The plant heights increased with
76
Potential Application of Kava (Piper methysticum F.) in Nematode Control
increasing concentrations for FJ kava but decreased with increasing concentration for VA
kava. Root length showed a slight decreasing trend for all experiments.
3.3.2.4 Six Weeks of Degradation
In this experiment, kava had been allowed to degrade in the soil for six weeks before the
seedlings were planted and inoculated. All of the three treatments were found to be
effective in controlling root knot nematodes. The number of root galls were inversely
related to the amount of kava added to soil. The control for FJ kava had an average
number of 17 galls while VA kava had 67 galls. Two plants in 2 % and three plants in 5
% concentration had died in FJ kava treatment while for VA kava treatment, one plant
each in 2 % and 5 % concentration had died. The general trend is similar in both FJ and
VA kava although in VA kava experiment, one plant had died in the control pots.
The plant height increased in 2 % and 1 % concentrations of FJ kava but a slight decrease
compared to control was seen in the 5 % treatment. All the treatment concentrations on
VA kava showed slight decreased plant heights. Root length measurements were lower
than control in all FJ kava treatments but for VA kava treatments lower values were
recorded for 1 % and 2 % concentrations while 5 % concentration showed slightly higher
value.
77
Potential Application of Kava (Piper methysticum F.) in Nematode Control
4
3 36
2
1
0
2 Weeks Degradation FJ Kava
% Kava in Soil
120 OT+->
100 J5Q.
80 >
60 to
40 0
+ 20
0
• % healthy plants (live plants) - Average of total number of Galls
Figure 3.16: Two weeks degradation FJ Kava
ling
e. g
al
<
161412
86420
2 Weeks Degradation VA Kava
% Kava in Soil
120
100 «
80 f60 |
40 S
20 $S
0
% healthy plants (live plants) . Average of total number of Galls
Figure 3.17: Two weeks degradation VA kava
78
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Gal
ling
Ave
.10 -i
987 -654321n
4 Weeks Degradation FJ Kava
x̂ :
r 120
100
80
60
40
20
n
1 2 5
% Kava in Soil
—•— % healthy plants (live plants ) A v e r a g e of total number of Galls
thy
Pla
nts
% H
eal
Figure 3.18: Four weeks degradation FJ kava
4 Weeks Degradation VA Kava
20
15
0
% Kava in Soil
100
80 I
60 S
40 |
20 ^
0
• % healthy plants (live plants) Average of total number of Galls
Figure 3.19: Four weeks degradation VA kava
79
Potential Application of Kava (Piper methysticum F.) in Nematode Control
gal
ling
Ave
.8 i
7 -654321r\U ~r
6 Weeks Degradation FJ
1 2
% Kava in Soil
—•— % healthy plants (live plants) —•—Average
Kava
j 120
" • CD
C
O
-̂o
o
oo
thy
Pla
nts
40 |
20 S?
5
of total number of Galls
Figure 3.20: Six weeks degradation FJ kava
6 Weeks Degradation VA Kava
35 n
30g> 25I 20° 15< 10
5
0
105
1 0 0c
95 £90 I"85 I80 S?
75
% Kava in Soil
% healthy plants (live plants ) Average of total number of Galls
Figure 3.21: Six weeks degradation VA Kava
80
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Table 3.14: Observed plant heights (cm) for the various treatment experiments of
FJ and VA kava
Concentration ofkava in soil (%)
0
1
2
5
FJ kava
0 wks
30.2
28.1
13.4
9.5
2 wks
35.3
43.3
40.4
36.9
4 wks
37.0
35.6
35.5
31.8
6 wks
26.5
31.3
33.8
24.6
VA kava
0 wks
28.1
22.6
20.2
20.0
2 wks
27.5
24.7
24.7
25.8
4 wks
32.3
30.5
27.1
25.6
6 wks
35.2
31.3
27.9
30.9
Table 3.15: Observed root lengths (cm) for the various treatment experiments of FJ
and VA kava
Concentrationof kava in soil
(%)
0
1
2
5
FJ kava
0 wks
16.6
29.7
16.4
14.5
2 wks
22.5
18.0
19.5
18.1
4 wks
21.3
17.4
19.5
19.6
6 wks
17.2
11.7
17.0
16.7
VA kava
0 wks
22.9
7.6
12.6
10.2
2 wks
23.3
17.8
18.8
16.8
4 wks
22.1
19.1
17.5
18.8
6 wks
17.4
16.5
15.2
18.7
81
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.2.5 Comparison of Observed Results at Different Concentrations over Various
Degradation Periods
Figures 3.22 and 3.23 summarize the results of the different degradation experiments of
FJ and VA kava at various concentrations tested. The number of galls observed at
different concentrations did not differ much from each other since the trend line increases
very slightly as the concentration is decreased. Statistical analysis shows that P < 0.001
(FFJ = 173.0, PFJ = 0.000, dfFJ = 1, 79; FVA = 152.2, PVA = 0.000, dfVA = 3, 73), which
indicates that concentration of kava was significant in the experiment. Comparison of the
different degradation periods over the concentration range does not show a very distinct
variation for FJ kava but is distinct for VA kava.
The trend lines for the different degradation periods of FJ kava get closer to each other as
the concentration is increased. There was significant interaction, P< 0.001 (FFJ = 27.4,
PFJ = 0.000, dfFJ = 9, 79) between the different concentrations and different degradation
periods. The 0 and 4 weeks degradation experiments gave similar results at the 1 %
concentration while the 2 and 6 weeks degradation showed slightly lower root gall
numbers but were close to each other. For VA kava the 2 weeks degradation experiment
was most effective in decreasing root gall numbers followed by 4, 0 and 6 weeks. The
observed P > 0.1 (FVA = 0.6, PVA = 0.74, dfVA = 9, 73) which indicates that there was no
significant interaction between the kava concentrations and the different degradation
periods for VA kava.
82
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Root Gall Numbers against Kava Concentration
1 2Kava Concentration in Soil (%)
0 weeks 2 2 weeks 4 weeks 6 weeks
Figure 3.22: Root gall numbers against kava concentration for FJ kava
Figure 3.23: Root gall numbers against kava concentration for VA kava
83
Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.3.2.6 General Discussion
Kava when used as soil amendment was effective in controlling root- knot nematode
population in all experiments. However some phytotoxic effect of kava was also noted
when it was used as soil amendment. In the 0 week degradation experiment, a number of
plants had died at the higher concentrations. Some plant mortality was also noted in the
other experiments at higher concentrations but was not very significant. Since whole kava
root material (powder) was used, any of the various compounds present in kava can be
responsible for these two (nematicidal and phytotoxic) activities. Some phenolic
compounds have already being isolated from kava which are also present in other plants
with allelopathic properties (Xuan et al., 20031).
Allelopathic properties are the ability to suppress plant growth, especially the growth of
troublesome weeds. Other chemicals identified in kava as reported by various authors
elsewhere could be responsible for these nematicidal effects (Cambie et al, 1997;Lebot
et al, 1992, 1997; O'hara et al. 1965; Singh and Blumenthal 1992, Singh 1986; Waiji
1998; Whitton et al, 2003; Xuan, et al, 20031).
Allowing kava to degrade in the soil before planting was seen to have lesser phytotoxic
effect compared to instant application such as at 0 week degradation. It was noted that
two weeks of degradation of kava in the soil was the most effective in suppressing root
galling while having lesser phytotoxic effect. No plant mortality was noted in the 2 weeks
degradation of FJ kava whilst some plant mortality was noted in VA kava. The difference
84
Potential Application of Kava (Piper methysticum F.) in Nematode Control
in the results for phytotoxicity for the 0 and 2 weeks degradation experiment indicates
that the compounds in kava responsible for this activity may have been destroyed
(degraded) or converted to harmless compounds over this period. In the case of VA kava,
it may not have been totally degraded in this time period.
The difference in properties of the two kava types (FJ and VA) could be due to either
different compounds or different amounts of the same compounds. If this activity is due
to different compound(s), then it can be said that these compounds were not broken down
during the degradation period or some other new compounds with phytotoxic effect may
have been synthesized from these chemicals. Alternatively it can also be possible that
higher concentration of the same compounds as that of FJ kava are present in VA kava
and it could not be totally degraded by the available microorganism population. Since
microorganism population was not determined in this study, these are only postulations
for such an activity. Microorganisms can degrade harmful substances to harmless ones
and can also produce new compounds from the existing compounds through in vitro
biochemical conversion through processes such as cyclisation and esterification. These
new compounds can be even more harmful than the initial compounds from which they
are synthesized (Begum et al, 2003; Halbrendt 1996; Rodriguez-Kabana 1986).
For the six weeks degradation experiment, it was noted that for VA kava the number of
plants dead in 2 % and 5 % concentrations were same as that in the control. The death of
a plant in the control would definitely be due to reasons other than the effect of kava. If
the plant mortality noted in the treatment pots are due to some other causes that is the
85
Potential Application of Kava (Piper methysticum F.) in Nematode Control
same cause as that for control, it can be said that VA kava after six weeks of degradation
does not have any phytotoxic effect. For FJ kava, plant mortality was noted in the six
weeks degradation experiment, while at 2 weeks of degradation no plant mortality was
seen. Hence it can be said that during this period new chemical compounds with
phytotoxic activity may have been generated from FJ kava by the microorganisms. The
nematicidal activity of kava was still prominent after six weeks of degradation. Thus kava
can keep nematodes in control for quite some time after it has been applied to soil.
The compounds in kava responsible for these activities can not be identified as yet since
chemical analysis of kava after various degradation periods was not carried out.
Generally all the experiments significantly decreased root gall numbers compared to the
controls but in some, plant growth or health was affected as mentioned above.
An important observation made during the degradation period of kava was that, the pots
in which kava had been added were usually dry. The dryness increased with increasing
concentration and required daily watering. Thus the dying of plants in the experiment, in
which no degradation was allowed, could be due to unavailability of water since water
added may have been absorbed by certain component(s) of kava. Allowing kava to
degrade in soil minimized this effect leading to healthier plants and decreased root galls.
This could possibly be due to degradation of the component(s) of kava which caused the
soil to dry. Chemical analysis of kava after different degradation period will only allow
the determination of the cause for this.
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Potential Application of Kava (Piper methysticum F.) in Nematode Control
3.4 CONCLUSION
The observations have revealed that kava does have the active ingredients to suppress the
root knot nematodes. Samples X as well as Y has shown positive results for nematode
control. These samples had initiated juvenile mortality within 24 hrs of exposure which
increased with increasing treatment times. The activity was permanent since none of the
test samples showed more than 50 % revival when exposed to distilled water after being
treated with the samples. Effective suppression of egg hatching was also noted for these
extracts. All the extracts tested showed less than 50 % hatching over the observation
period.
The results are also significant as the dosage found to be effective against these
nematodes is quite low. Concentration as low as 2 % (w/v) showed significant juvenile
mortality and effective suppression of egg hatch. The isolated fractions of samples Y-FJ
and Y-VA were also effective towards juvenile mortality but recorded percentage
mortality was lower than samples X and Y. It could have been possibly due to the low
concentration (0.1 % w/v) at which these samples were tested. Since both sample X as
well as Y (sample X less ethanol solubles) showed nematicidal activity, it can be
concluded that the nematicidal activity of kava was not entirely due to groups of
compounds with similar chemistry but the effect of the various compounds together. That
is the result of the group of highly polar compounds present in sample Y as well as that of
the ethanol solubles.
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Potential Application of Kava (Piper methysticum F.) in Nematode Control
The pot experiments have shown that mixing kava powder in soil before planting can
help in reducing infection by the root knot nematodes. These experiments have indicated
that kava should be allowed to degrade in the soil for at least two weeks before planting
to eliminate the phytotoxic effect. It was noticed that when seedlings were planted in the
soil on the day of addition of kava powder, a high number of plants were dead. While the
two, four and six weeks of degradation periods for both FJ and VA kava showed lesser
affect on the health of the plant. It was also noted that the active ingredients in kava
responsible for minimizing root gall numbers were effective even after 6 weeks since root
galls were suppressed in the 6 weeks degradation experiments as well. Therefore using
kava as soil amendment for controlling RKN would not require frequent re-application.
Kava has been found to be quite effective in these experiments. The extracts tested can be
further fractionated and re-tested to identify the compound(s) with nematicidal activity.
Once the active compound has been identified, large scale isolations can been done to use
kava as a nematicide. Moreover, with these initial positive results of kava, further
exploration can be carried out in unused portions of kava plant such as the peelings and
leaves as well as in related and wild kava species. Chemical analysis of kava after
different degradation periods would permit determination of the different compounds
present after the various degradation periods. This will assist in identifying the phytotoxic
compounds in kava. Therefore these compounds which can be removed from kava when
incorporating kava extracts for nematode control at large scale.
88
Potential Application of Kava (Piper methysticum F.) in Nematode Control
CHAPTER 4
4.0 GENERAL CONCLUSIONS
• The water soluble components as in the samples X of FJ and VA kava can be said
to contain different compounds or different amounts of the same compound. This
is because samples X of the two kava varieties showed different effect on the
root-knot nematode juveniles. While FJ kava showed continued mortality, VA
kava demonstrated some reversible of the initial effect noted when the juveniles
were transferred to distilled water.
• Samples Y of both kava types and their corresponding fractions could possibly
contain similar chemicals since they showed similar activity on juveniles but
further analysis needs to be done to confirm this.
• Samples X showed stronger nematicidal activity compared to sample Y for both
FJ and VA kava. This indicates that the ethanol solubles which had been removed
while preparing sample Y had significant effect on the root-knot nematodes.
• Both samples X and Y of FJ as well as VA kava were equally effective in
suppressing egg hatching. It can be said that the juvenile mortality and
suppression of egg hatching was due to different compounds in kava. However, it
89
Potential Application of Kava (Piper methysticum F.) in Nematode Control
could also be due to the same compounds since the effect of some chemicals on
different stages of an organisms life cycle are different.
Treatments that were replenished with distilled water on the 3rd day of observation
seemed to record slightly more hatched juveniles on the 7th day compared to the
treatments where the test solutions were added. It was noticed that addition of
water dilutes the effect of the test solutions thereby allowing more hatching.
• The pot experiments showed that a minimum of 2 weeks of degradation
eliminates/minimizes the phytotoxic effect of kava.
• Glucosides (in addition to carbohydrates) have been postulated to be present in
samples Y from the H-NMR data. A number of glucosides are known to have
nematicidal properties. Hence the glucosides in kava could be responsible for the
nematicidal activity. Moreover phenolic compounds present in kava have been
isolated from other plants and been tested positive for nematicidal properties as
well.
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Potential Application of Kava (Piper methysticum F.) in Nematode Control
CHAPTER 5
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APPENDICES
Appendix 1
Flow Diagram outlining the extraction procedure for Kava
Powdered Kava Root Material
Residue
H2O
Residue(Sample Y)
Aqueous Extract
Freeze Dry
Residue(Sample X)
Extraction withEthanol
Ethanol Extract
Semi-preparative HPLCfractionation
SampleY1
SampleY2
SampleY3
SampleY4
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Potential Application of Kava (Piper methysticum F.) in Nematode Control
Appendix 2
^ Spectrum of Sample Yl
i
I — ^
106
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Spectrum of Sample Y2
107
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Spectrum of Sample Y3
108
Potential Application of Kava (Piper methysticum F.) in Nematode Control
Spectrum of Sample Y4
109