THE YIELD AND BIOLOGICAL ACTIVITY (LC50) OF ROTENONE

EXTRACTED FROM Derris elliptica

SAIFUL IRWAN BIN ZUBAIRI

A thesis submitted in fulfilment of the

requirements for the award of the degree of

Master of Engineering (Bioprocess)

Faculty of Chemical and Natural Resources Engineering

Universiti Teknologi Malaysia

AUGUST 2006

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To my most beloved sayang and mak

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ACKNOWLEDGEMENTS

‘In the name of Allah, the most gracious and the most merciful. Selawat and

salam to Prophet Muhammad s.a.w.’ I owe first and foremost my profound gratitude to

almighty Allah s.w.t the source of all inspiration and help and without whose assistance;

this study would not have come into existence.

Deep obligation and indebtedness and most sincere gratitude are offered to my

supervisor Professor Dr. Mohammad Roji Sarmidi for his continuous guidance during

all stages of my research work and for his willingness to help. Without his continue

support, interest, wisdom and idea during our discussion, this thesis would not have been

implemented and executed well.

I would also like to acknowledge the Chemical Engineering Pilot Plant (CEPP)

staff, in particular Professor Ramlan Abdul Aziz for his moral support and motivation as

well as Mr. Khairul Annuar Mohd, Ms. Nor Idamalina Ahamad Nordin and Mr. Rafizan

Latip for their assistance on the batch solid-liquid extraction process, qualitative analysis

of Vacuum Liquid Chromatography-Thin Layer Chromatography (VLC-TLC) and

biological activity (LC50) of rotenoids resin.

Last but not least, I would like to express my heartfelt gratitude to my most

beloved parents and wife, Allahyarham Zubairi Abdul Wahid, Zaiton A. Man and

Nurhafzan Anis Ismail respectively, through which the guidance of the spiritual, mental

and physical training that has allowed me to established and continue throughout this

study.

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ABSTRACT

The objective of this research was to determine the effect of the processing parameters

on the extraction yield of rotenoids resin, rotenone and their biological activities (LC50).

The research was divided into three stages: preliminary, optimization and verification

phases. Preliminary study was carried out to determine the most appropriate processing

parameters for the optimization study. The optimization study was carried out using a

Central Composite Design (CCD) employing the Design-Expert® software version 6.0 to

determine the effects of processing parameters on the three selected response variables

which were the yield of rotenoids resin, yield of rotenone and biological activity (LC50)

of rotenoids resin. The processing parameters studied were the types of solvent

(acetone, chloroform and ethanol), solvent-to-solid ratio (2.0 ml/g to 10.0 ml/g) and raw

material particles size (0.5 mm to 5.0 mm in diameter). The theoretical maximum yield

of rotenoids resin in dried roots obtained from the optimization phase was 12.26 %

(w/w) and 5.99 % (w/w) for the rotenone. The multiple response variables analysis have

consistently verified the theoretical results in the range of 3.62 ml/g to 4.72 ml/g

(solvent-to-solid ratio) and 0.83 mm to 1.41 mm in diameter (raw material particles size)

using the acetone extract. The biological activity (LC50) value of rotenoids resin was

indirectly correlated to the optimum processing parameters due to inconsistency of

rotenone content (mg) and the low value of LC50 which was less than 100 ppm for all

treatments. This is due to the presence of other constituents in the rotenoids resin

(tephrosin, 12αβ-rotenolone and deguelin) which contributed to the low LC50 values.

The optimization of the processing parameters resulted in an increase of yield of

rotenoids resin but reduced yield of rotenone.

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ABSTRAK

Objektif kajian ini adalah untuk menilai kesan parameter pemprosesan terhadap

pengekstrakan keberhasilan resin rotenoids, rotenone dan aktiviti biologikalnya (LC50).

Kajian ini dibahagikan kepada tiga peringkat: fasa saringan, pengoptimuman dan

penentusahan. Kajian saringan dijalankan untuk menentukan parameter pemprosesan

yang paling relevan untuk kajian pengoptimuman. Kajian pengoptimuman dijalankan

menggunakan analisis ‘Central Composite Design (CCD)’ menggunakan perisian

‘Design-Expert® version 6.0’ bagi menilai kesan parameter pemprosesan bagi tiga

variabel respon yang dipilih iaitu keberhasilan resin rotenoids, keberhasilan rotenone

dan aktiviti biologikal (LC50) bagi resin rotenoids. Parameter pemprosesan yang dikaji

adalah jenis pelarut (aseton, kloroform dan etanol), nisbah pelarut terhadap pepejal (2.0

ml/g hingga 10.0 ml/g) dan saiz partikel bahan mentah (0.5 mm hingga 5.0 mm dalam

diameter). Keberhasilan maksimum teori resin rotenoids di dalam akar kering yang

diperolehi daripada fasa pengoptimuman adalah 12.26 % (w/w) dan 5.99 % (w/w) untuk

rotenone. Analisis kepelbagaian variabel respon mengesahkan secara konsisten

keputusan teori di dalam julat 3.62 ml/g hingga 4.72 ml/g dan 0.83 mm hingga 1.41 mm

dalam diameter menggunakan pengekstrakan aseton. Nilai aktiviti biologikal (LC50)

resin rotenoids tidak berkaitan secara langsung dengan parameter pemprosesan optimum

disebabkan oleh kandungan rotenone (mg) yang tidak konsisten dan nilai LC50 yang

rendah di mana kurang daripada 100 ppm bagi semua rawatan. Ini disebabkan oleh

kewujudan kandungan lain di dalam resin rotenoids (tephrosin, 12αβ-rotenolone dan

deguelin) di mana turut menyumbang kepada nilai LC50 yang rendah. Pengoptimuman

parameter pemprosesan di dapati telah menyebabkan peningkatan keberhasilan resin

rotenoids tetapi mengurangkan keberhasilan rotenone.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE PAGE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF FIGURES xiv

LIST OF TABLES xix

LIST OF ABBREVIATIONS xxii

LIST OF APPENDICES xxiv

1 INTRODUCTION

1.1 Research background 1

1.2 Scopes of research 7

1.3 Contribution of the research 8

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2 TUBA, Derris elliptica: OVERVIEW, BIOLOGY,

CULTIVATION AND PHYTOCHEMISTRY

2.1 Overview of the phytochemicals 9

2.1.1 Metabolic pathway of the

phytochemical insecticides 10

2.2 Derris elliptica or ‘Tuba’ 12

2.3 Scientific classification (taxonomy) and species 13

2.3.1 Plant growth, development and ecology 13

2.3.2 The cultivation condition of Derris elliptica 14

2.3.3 Current development on the cultivation

of Derris elliptica 16

2.4 Phytochemistry of Derris species 16

2.4.1 Outline of rotenone as an active chemical

constituents 16

2.4.2 Physico-chemical properties of rotenoids 17

2.4.3 Rotenone stability in water 20

2.4.4 Rotenone stability in soil and groundwater 21

2.4.5 Rotenone stability in vegetation 22

2.4.6 Types of rotenone formulation 22

3 PROCESSING, ANALYSIS AND TOXICOLOGY

3.1 Introduction 24

3.1.1 Extraction method 25

3.2 Extraction mechanism 26

3.2.1 Principles of solid-liquid extraction 28

3.2.1.1 Types of solid-liquid extraction 28

3.2.1.2 Desirable features for the

extracting solvent 28

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3.2.1.3 Leaching process

(solid-liquid extraction) 29

3.2.2 Extraction of the rotenone and rotenoids

resin: An overview of pilot and industrial

plant scale production 30

3.3 Analytical methods 34

3.4 Toxicology 34

3.4.1 The use of biological assays to

evaluate botanicals 34

3.4.1.1 Dose-response curves 36

3.4.1.2 Hazard indicator categories 38

3.4.1.3 Toxicity assessment by probit

analysis 40

3.4.2 Brine Shrimp (Artemia salina)

Lethality study 41

3.4.2.1 Artemia life history 41

3.4.2.2 Hatching the Artemia 41

3.4.2.3 Harvesting the nauplii 42

3.4.2.4 Maintenance of brine shrimp 43

3.4.2.5 Optimum Artemia survival

condition 44

3.4.3 Rotenone toxicology data 45

3.4.3.1 Mode of action 45

3.4.3.2 Toxicity 45

(a) Human data 45

(b) Aquatic life data 46

(c) Relevant animal data 46

(c) Relevant in vitro data 47

(d) Workplace standards 47

(e) Acceptable Daily

Intake (ADI) 47

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(f) Carcinogenicity 47

(g) Mutagenicity 47

(h) Interactions 47

3.4.4 CASE STUDY: Laboratory and field

efficacy studies on the toxicity of the

formulated rotenone 48

3.4.4.1 Laboratory studies (bioassay) 48

3.4.4.2 Field efficacy studies 50

4 METHODOLOGY

4.1 Introduction 51

4.1.1 Preliminary experiments 52

4.1.2 Optimization phase 53

4.1.2.1 Design of Experiments (DOE) 54

4.1.2.2 Factors and experimental matrix 55

4.1.3 Verification phase 58

4.2 Sampling 58

4.3 Process description 58

4.3.1 Pre-processing of Derris roots 60

4.3.2 Extraction of rotenoids resin 61

4.3.3 Analysis of the response variables 63

4.3.3.1 Determination of extraction yield

(rotenoids resin) 63

4.3.3.2 Determination of extraction yield

(yield of rotenone) 64

(a) Qualitative analysis of

rotenone using Thin Layer

Chromatography (TLC) 64

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(b) Quantitative analysis of

rotenone using High

Performance Liquid

Chromatography (HPLC):

Measurement of the

rotenone content (mg) 65

(c) Biological activity (LC50)

of rotenoids resin 67

4.4 Statistical analysis 69

4.4.1 Response Surface Methodology (RSM) 70

4.4.2 Model adequacy checking 71

4.4.2.1 F-distribution test 71

4.4.2.2 Coefficient of multiple

determinations (R2) 72

4.4.2.3 Lack of fits test 72

4.4.3 Pearson’s correlation coefficient, r 73

5 RESULT AND DISCUSSION

5.1 Introduction 74

5.2 Preliminary experiment results 75

5.2.1 Effects of the plant parts and types

of solvent on yield 75

5.2.2 Extraction yield model and the effect

of extraction duration on yield 77

5.2.3 Effects of the extraction and concentration

operating temperature on yield 82

5.2.4 Effect of the raw material particles size

and solvent-to-solid ratio on yield 86

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5.2.5 Summary of the preliminary experiments 94

5.3 Optimization phase results:

Effect of processing parameters on the response

variables 98

5.3.1 Effect of processing parameters on the

yield of rotenoids resin in dried roots 100

5.3.2 Effect of processing parameters on the

yield of rotenone in dried roots 109

5.3.3 Summary of the optimization phase 118

5.4 Multi response analysis of the yield of rotenone

in dried roots; % (w/w) and rotenone concentration

(mg/ml) 120

5.4.1 Analysis of solvent-to-solid ratio (ml/g)

for the ethanol + oxalic acid solution

extract in relation with the yield of

rotenone in dried roots; % (w/w) and

rotenone concentration; mg/ml 121

5.4.2 Analysis of solvent-to-solid ratio (ml/g)

for the acetone extract in relation with

the yield of rotenone in dried roots;

% (w/w) and rotenone concentration; mg/ml 122

5.4.3 Analysis of raw material particles size

(mm in diameter) for the ethanol +

oxalic acid solution extract in relation

with the yield of rotenone in dried roots;

% (w/w) and rotenone concentration; mg/ml 125

5.4.4 Analysis of raw material particles size

(mm in diameter) for the acetone extract

in relation with the yield of rotenone in

dried roots; % (w/w) and rotenone

concentration; mg/ml 127

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5.5 Biological activity (LC50) of rotenoids resin results 129

5.5.1 The effect of raw material particles size

and types of solvent on the biological

activity (LC50) and yield of rotenone

in dried roots, % (w/w) respectively 132

5.5.2 The effect of solvent-to-solid ratio

and types of solvent on the biological

activity (LC50) and yield of rotenone

in dried roots, % (w/w) respectively 135

5.5.3 Biological activity (LC50) of the

verification phase parameters and

rotenone standard (SIGMA-Aldrich™) 143

5.6 Verification phase results:

Confirmation of the optimization 143

5.7 Comparison of the optimum response variables 144

5.8 Correlation between the yield of rotenoids

resin and yield of rotenone 148

6 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions 151

6.3 Recommendations 154

REFERENCES 155

APPENDICES 173

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LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 Biosynthetic origin of some commercially important plant-derived compounds 11 2.2 Derris species that abundantly available in Peninsular of Malaysia: (A) Derris elliptica and (B) Derris malaccensis 13 2.3 Rotenone molecular structures 18 3.1 Schematic diagram of vegetal cell structures 27 3.2 Layout of the pilot plant scale production of the Concentrated Liquid Crude Extract (CLCE) 32 3.3 Dose-response curve 36 3.4 An adult of Artemia salina: (A) male; (B) female 41 3.5 Example of the Brine Shrimp hatchery system 44 3.6 The leaf-dipped method 49 3.7 The larvae of diamondback moth (Plutella xylostella) 49 3.8 Field efficacy of formulated rotenone against Spodotera litura 50 4.1 Phases of the experiment 59 4.2 Flow diagram and overview of the study 59 4.3 Various particles size of Derris roots 60

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4.4 Extraction of rotenoids resin from Derris elliptica roots 62 4.5 Evaluation of rotenoids resin 63 4.6 Techniques of spotting the sample on silica plate 65 4.7 External standard method template calculations 66 4.8 Example of the dilution principles to prepare the biological activity concentration 68 4.9 Mortality of Artemia salina when exposed to extracts of the Derris elliptica: (A) dose response curve; (B) probit analysis curve 69 5.1 Yield of rotenone in dried roots, % (w/w) using the Normal Soaking Extraction (NSE) method for different types of solvent 76 5.2 Kinetic equilibrium of the rotenone extraction process (second order polynomial) 80 5.3 Kinetic equilibrium of the rotenone extraction process: Yield of rotenone content in dried roots, % (w/w) 81 5.4 Kinetic equilibrium of the rotenone extraction process: Concentration of rotenone, mg/ml 81 5.5 Kinetic equilibrium of the rotenone extraction process: Yield of rotenone in dried roots, mg 82 5.6 Degradation of rotenone content (mg) during the concentration process at 40 0C and 0.3 mbar of operating temperature and vacuum pressure respectively 83 5.7 Degradation of rotenone content (mg) during the concentration process at 50 0C and 80 mbar of operating temperature and vacuum pressure respectively 84 5.8 Kinetics of the rotenone extraction process from

Derris elliptica - Ethanol + oxalic acid solution: (A) rotenone concentration, mg/ml; (B) yield of rotenone, % (w/w) 85

5.9 Kinetics of the rotenone extraction process from Derris elliptica - Chloroform: (A) rotenone concentration, mg/ml; (B) yield of rotenone, % (w/w) 90

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5.10 Kinetics of the rotenone extraction process from Derris elliptica - Acetone: (A) rotenone concentration, mg/ml; (B) yield of rotenone, % (w/w) 92

5.11 Response surface three-dimensional graphs and contour plot 99 5.12 Normal probability plots of residuals (Yield of rotenoids resin) 104 5.13 The residual versus the predicted response (Yield of rotenoids resin) 104 5.14 Surface plot of the yield of rotenoids resin in dried roots, % (w/w) as a function of raw material particles size and solvent-to-solid ratio: Ethanol + oxalic acid solution extract 105 5.15 Surface plot of the yield of rotenoids resin in dried roots, % (w/w) as a function of raw material particles size and solvent-to-solid ratio: Acetone extract 105 5.16 Normal probability plots of residuals (Yield of rotenone) 113 5.17 The residual versus the predicted response

(Yield of rotenone) 114

5.18 Surface plot of the yield of rotenone in dried roots, % (w/w) as a function of raw material particles size and solvent-to-solid ratio: Ethanol + oxalic acid solution extract 114 5.19 Surface plot of the yield of rotenone in dried roots, % (w/w) as a function of raw material particles size and solvent-to-solid ratio: Acetone extract 115 5.20 Selected processing parameters that obtain maximum yield of rotenoids resin in dried roots, % (w/w) and yield of rotenone in dried roots, % (w/w) based on the desirability values of a given solution 120 5.21 The yield of rotenone in dried roots; % (w/w) and rotenone concentration; mg/ml versus the solvent-to-solid ratio (ml/g) of ethanol + oxalic acid solution extract 123

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5.22 The yield of rotenone in dried roots; % (w/w) and rotenone concentration; mg/ml versus the solvent-to-solid ratio (ml/g) of acetone extract 125 5.23 The yield of rotenone in dried roots; % (w/w) and rotenone concentration; mg/ml versus the raw material particles size (mm in diameter) of ethanol + oxalic acid solution extract 127 5.24 The yield of rotenone in dried roots; % (w/w) and rotenone concentration; mg/ml versus the raw material particles size (mm in diameter) of acetone extract 129 5.25 Relationship between the probit of Artemia salina mortality proportion and log10 dose of rotenoids resin (S1) at 24 hours of treatment 130 5.26 Relationship between the probit of Artemia salina mortality proportion and log10 dose of the rotenoids resin (S1, S7, S20 and S23) at 12 hours of treatment 130 5.27 Relationship between the probit of Artemia salina mortality proportion and log10 dose of rotenoids resin (S7, S11, S13, S19, S23, S24, S25, S28 and S29) at 12 hours of treatment: Continued 131 5.28 Relationship between the probit of Artemia salina mortality proportion and log10 dose of rotenoids resin (S3, S8 and S12) at 6 hours of treatment 132 5.29 Effect of the raw material particles size, mm in diameter

against the biological activity (LC50) of acetone extract (A1) and ethanol + oxalic acid solution extract (A2) respectively 133

5.30 Effect of the raw material particles size, mm in diameter against the yield of rotenone in dried roots, % (w/w) obtained from the extract of acetone (B1) and ethanol + oxalic acid solution (B2) respectively 135

5.31 Effect of the solvent-to-solid ratio, ml/g against the biological activity (LC50) of acetone extract (C1) and ethanol + oxalic acid solution extract (C2) respectively 137 5.32 Effect of the solvent-to-solid ratio, ml/g against the yield of rotenone in dried roots, % (w/w) obtained from the extract of acetone (D1) and ethanol + oxalic acid solution (D2) respectively 139

xviii

5.33 Pearson’s correlation coefficients (r) between the yield of rotenone in dried roots; % (w/w) and yield of rotenoids resin in dried roots; % (w/w) 149

xix

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Existence of rotenone in Derris elliptica Benth plant’s organs determined by different analysis methods 15 2.2 Rotenone, methionine and phenylalanine in different organs of Derris plant analyzed by reversed-phase HPLC 15 2.3 The solubility of pure rotenone at 20 0C 19 2.4 Time of rotenone dissipation versus temperature 20 2.5 Detoxification time of varies rotenone concentration 21 3.1 Rotenone extraction methods 33 3.2 Hazard indicator categories 39 3.3 Toxicity of the botanical insecticides against the larvae of DBM collected from Kluang, Johor 48 3.8 Toxicity of the botanical insecticides against the larvae of

DBM collected from Karak, Pahang 49

4.1 Preliminary processing parameters 52 4.2 The preliminary experiment to obtain the rotenoids resin based on the exploratory experiment carried out by Saiful et al. (2003) 52 4.3 Preliminary control processing parameters 53 4.4 Experimental design for the solvent-to-solid ratio of 3.3 ml/g 53

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4.5 Experimental design for the solvent-to-solid ratio of 10.0 ml/g 53 4.6 Preliminary response variables 53 4.7 Specification of Central Composite Design (CCD) 54 4.8 Optimization processing parameters 55 4.9 Optimization control processing parameters 56 4.10 Optimization response variables 56 4.11 Experimental matrix for the extraction of rotenoids resin: CCD (23) 57 4.12 Parameters of RP-HPLC recommended by Baron and Freudenthal (1976) 66 5.1 Processing parameters involved in the kinetic of rotenone extraction process 79 5.2 Response variables result in the kinetic of rotenone extraction process 80 5.3 The average yield of rotenone in dried roots, % (w/w) 95 5.4 The preliminary experiments result 95 5.5 The design layout and experimental results

(Yield of rotenoids resin) 100

5.6 ANOVA response surface linear model [responses: Yield of rotenoids resin in dried roots, % (w/w)] 101

5.7 The design layout and experimental results (Yield of rotenone) 109

5.8 ANOVA response surface 2FI model (responses: Yield of rotenone in dried roots) (backward) 110 5.9 Selection criteria of the processing parameters solution 119 5.10 The effects of solvent-to-solid ratio (ml/g) of ethanol + oxalic acid solution extract on the two response variables 122

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5.11 The effects of solvent-to-solid ratio (ml/g) of acetone Extract on the two response variables 124 5.12 The effects of raw material particles size (mm in diameter) of ethanol + oxalic acid solution extract on the two response variables 126 5.13 The effects of raw material particles size (mm in diameter) of acetone extract on the two response variables 128 5.14 Biological activity (LC50) of rotenoids resin at varies time of treatment (6 hours, 12 hours and 24 hours) 140 5.15 Effect of rotenoids resin against Artemia salina at varies time of treatment (6 hours, 12 hours and 24 hours) 141 5.16 Effect of rotenoids resin against Artemia salina on the 24 hours of treatment established by McLaughlin (1991) 142 5.17 List of selected processing parameter that produced theoretical maximum yield of rotenoids resin in dried roots; % (w/w) and yield of rotenone in dried roots, % (w/w) 143 5.18 The verification phase results based on the most appropriate processing parameters 144 5.19 Comparison of the optimum response variables with Different phases of experiment 147 5.20 Pearson’s correlation coefficients (r) of the response variables 148

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LIST OF ABBREVIATIONS

ANOVA - Analysis of Variance

A.i - Active ingredient

CEPP - Chemical Engineering Pilot Plant

CCD - Central Composite Design

CLCE - Concentrated Liquid Crude Extract

CP - Centre point

DAT - Days after treatment

DBM - Diamondback Moth

DF - Dilution factor

DIW - Deionized water

DOE - Design of Experiments

EPA - Environmental Protection Agency

EC - Emulsifiable Concentrates

IS - Internal standard

IPM - Integrated Pest Management

Kg - Kilogram

LC50 - Lethal Concentration of 50 % mortality

LD50 - Lethal Dose of 50 % mortality

LCE - Liquid Crude Extract

L - Litre

Ibm - Pound-mass

NSE - Normal Soaking Extraction

NPK - Nitrogen, Phosphorus and Kalium

ND - Not determined

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m.p - Melting point

OA - Oxalic acid

ppm - Part per millions

PDA - Photo Diode Array

Rf - Retardation factor

RSM - Response Surface Methodology

RP-HPLC - Reversed-Phase High Performance Liquid Chromatography

RCBD - Randomized Complete Block Design

SFE - Supercritical Fluid Extraction

SST - Total of Sum of Squares

SSR - Sum of Squares due to Regression

SSE - Sum of Squares of Residuals

SF - Sensitivity factor

SF - Safety factor

SD - Standard deviation

SG - Specific gravity

SHD - Safe Human Dose

TLC - Thin Layer Chromatography

ThD0.0 - Threshold Dose

UTM - Universiti Teknologi Malaysia

UPM - Universiti Putra Malaysia

UV - Ultra Violet

VLC - Vacuum Liquid Chromatography

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LIST OF APPENDICES

APPENDIX TITLE PAGE

A Complete results of the optimization phase experimental design 174 B Mortality of Artemia salina against varies

concentration of the rotenone Concentrated Liquid Crude Extract (CLCE) 179

C Experimental design and results (experimental and predicted values) of Central Composite Design; CCD (Manual calculation) 184 D The upper critical values of the F-distribution for v1 numerator degrees of freedom and v2 denominator degrees of freedom 5 % significance level: F0.05 (v1, v2) 190 E The biological activity (LC50) of S1 (24 hours of treatment) using probit analysis (manual calculation) 192 F Mortality of Artemia salina against varies

concentration (ppm) of rotenone Concentrated Liquid Crude Extract (CLCE) and rotenone standard (SIGMA-Aldrich™) 194

G Complete results of the verification phase and preliminary experiment (yield of rotenoids resin) 196 H Chromatograms of rotenone standard

[SIGMA-Aldrich™, 95 - 98 % (w/w)] and Sample 20 (S20) for the LCE and CLCE 198

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I Purification and identification of rotenone from Derris elliptica using the Vacuum Liquid Chromatography-Thin Layer Chromatography (VLC-TLC) method 201 J Molecular structure of 12αβ-rotenolone, tephrosin and deguelin 206

CHAPTER I

INTRODUCTION

1.1 Research background

One of the important issues facing approximately 6.48 billions world populations

(Anonymous, 2005) is food security. The over population in developing countries and

low food production exacerbated the situation. Low food production productivity is due

to many factors. One of the factors is due to pest and plant diseases.

Crop protections today rely heavily on synthetic pesticides (Coats, 1994). Their

uninterrupted and massive use has led to several side effects such as pesticides resistance

in pests (Stoll, 1988), elimination of naturally occurring bio control agents, insect

resurgence, adverse effects on non-target organisms and environment contaminations

with the potential effect on the entire food chain (Copping, 1998; Harris, 1999). The

growing public alarm about the hazards associated with excessive use of synthetic

pesticides has revived the interest in the use of environmental-friendly crop protection

products or well known as phytochemical pesticides. Phytochemical pesticides are

considered environmentally benign, biodegradable (Devlin and Zettel, 1999), maintain

2

biological diversity of predators (Grainge and Ahmad, 1998) and safer to higher animals

including human beings. Thus, to help meet the food requirements of the 21st century,

scientist throughout the world is looking for ecologically safe plant protection

technologies emphasizing use of the botanical insecticides in the integrated pest

management (IPM) programmes.

A vast number of plant species produce phytochemicals that are not directly

beneficial for the growth and development of the plants. These secondary compounds

are regarded as a part of the plants defence against plant-feeding insects and other

herbivores (Dev and Koul, 1997). The pesticide properties of many plants have been

known for a long time and natural pesticides based on plant extracts such as rotenone,

nicotine and pyrethrum have been commonly used in pest control during the earlier half

of this century. However, after the World War II, they lost their importance with the

introduction of the synthetic organic chemicals (Suraphon and Manthana, 2001). The

synthetic organic chemicals were concentrated products with a high knockdown effect

on pest organisms. These chemicals could be produced in large quantities at relatively

lower cost and they rapidly substituted most of the other pesticides (especially natural

pesticides) in the 1950s. However, with the development of resistant insects, the threat

of contaminated food and high production cost problems, natural pesticides came back

again in 1995 (Suraphon and Manthana, 2001).

Furthermore, Malaysia is one of the most diverse countries to have plants and

animals in the Southeast Asia region. Some of the plants show ability to adapt to and

tolerate herbivores and their environment. The adapting ability derived from the

production of special chemicals is called allelochemicals, which are parts of the

secondary plant substances (Yu and Hsu, 1985). Plant active ingredient that shows

hormonal inhibition in insects is such as azadiracthin from neem seed kernels. The other

that show repellent property is such as citronella from citrus leaves (Visetson and Milne,

2001). In general, plants with pesticidal properties can be used in three ways. Initially,

from the whole plant parts, in powder form or as crude extracts in water or other

solvents. Secondly, as purified extracts, such as rotenone and finally as a chemical

3

template which could be produced industrially such as pyrethrins (chemically

synthesized). Moreover, natural insecticides have been used for centuries to combat

insect’s pests that compete for food and affect the public health (WHO, 1997). As for

that reason, more than 2,000 plant species are catalogued as having insecticide

properties (Heal et al., 1950; Farnsworth, 1966; Sukamar et al., 1991). The most

commonly plant extracts such as pyrethrum, nicotine, saponin and rotenone were among

the first compounds used to control insects of agricultural importance (Coasts, 1994;

Grainage and Ahmed, 1998). Among the ordinary plant extracts that have been studied

and commercialized is pyrethrin (which a complex of esters extracted from flowers of

Chrysanthemum cinerariefolium) which is the one that still be used nowadays to

enhance commercial preparations of the household insecticides (Bell et al., 1990).

Moreover, nicotine extracted from Nicotiana glauca and its nicotinoids derivatives are

also among the choice molecules for the manufacture of new insecticides.

In addition, the one and only plant extracts that in the route of developing its

niche market to be among the important insecticides is rotenone. In general, rotenone

and other toxic constituents in the extract (e.g.: deguelin, tephrosin and 12αβ-

rotenolone) are isoflavonoids occurring in several genera of tropical leguminosae plants

such as Derris (papilionaceae), Antonia (loganiaceae) and Lonchocarpus (fabaceae).

Scientifically, rotenone is a bio-active compound that has a strong paralysis action

(knock-down effect) on cold-blooded animals and used as an insecticide to combat pests

(John, 1944; Andel 2000). Other than that, rotenoids-yielding plants have been also

used for fishing based on their itcthyotoxic properties (Andel, 2000). For that reason,

there have been reports of rotenone-containing plants used by the Indians for fishing due

to its itcthyotoxic effect, as early as the 17th century (Moretti and Grenard, 1982).

Interestingly, rotenone poisoned fish is edible without any risk of food contamination to

humans (Costa et al., 1989). The risk of being poisoned by rotenone on mammalians

can be strongly justify with the lethality tests resulted in LD50 (value is in milligram per

kilogram (mg/kg) of body weight in mammal) range from 50 to 300 (Raws, 1986;

Ellenhorn and Barceloux, 1988) and considered as a moderate hazardous substances

(WHO, 1992).

4

As for it lethal mechanism, rotenone acts by inhibiting respiratory enzyme in the

insects resulting in disruption of cellular metabolism and failure of respiratory functions

(Oberg, 1961; Fukami et al., 1967; Bradbury, 1986). Although rotenone is toxic to the

nervous systems of insects, fish and birds, commercial rotenone products presented little

hazard to humans over many decades (Schmeltz, 1971). Neither fatalities nor systemic

poisonings in humans have been reported in relation to ordinary use. As for that reason,

human or mammals are not highly susceptible or vulnerable to rotenone because they are

protected by effective oxidizing enzyme systems (Schnick, 1974) and inefficient

gastrointestinal absorption (Bradbury, 1986). This extensive research and thorough

evidence on its effect against targeted organisms and non-targeted organisms (especially

human) gives rotenone as one of the botanical insecticide that exceptionally selective

and environmental-friendly (Schnick, 1974; Bradbury, 1986). Other reasons that

accounting safety record of rotenone as a botanical insecticide are low concentration in

commercial products, highly degradable and poor absorption across gut and skin of

humans. Even though rotenone is a naturally occurring chemical with insecticidal,

acaricidal (mite and spider killing) and piscicidal (fish-killing) properties (Extoxnet,

1996), it is a selective, non-specific insecticide and also can be used in home gardens

for insect control, for lice and tick control on pets and for fish eradications as part of

water body management (Weier and Starr, 1950). Because of its advantages, the

extracts material can be formulated into emulsifiable concentrates (EC) and wettable

powders of rotenone and extensively used in lakes, ponds and reservoirs to control

undesirable fish as well as to combat the highly resistant insect pests that still posses a

major threat to farmers all over the world (Kole et al., 1992).

Nowadays, the production of botanical insecticide especially rotenone (from

Derris and Lonchocarpus species) and pyrethrum (from Chrysanthemum

cinerariaefolium) are dominated by the Western country such as Germany, United States

(US), Canada and South America (Murray, 1997). They have the technology to extract,

formulate and purify the bio-active constituents from plant material that have the

insecticidal properties. One of the biggest manufacturers in Europe that produced the

formulated liquid emulsion of rotenone and cube rotenoids resin is SAPHYR S. A. R. L

5

which is based in France. According to Grinda et al. (1986), the used on a batch solid-

liquid of Accelerated Solvent Extraction (ASE) method has made them produced as

much as 14 % (w/w) yield of rotenone in dried roots and 36 % (w/w) yield of rotenoids

resin in dried roots. This achievement is due to the advanced processing techniques they

have implemented and the usage of strong chlorinated organic solvent that extract the

bio-active constituents exhaustively. They have the technological advantages as

compared to the other countries (especially in Asia) that also produced botanical

insecticides product.

In Asia, only several countries are committed on developing the technology and

pursuing to produce large scale of botanical insecticide such as Thailand and Vietnam.

According to Hao et al. (1998), in Vietnam, they have conducted and set up a technical

process to manufacture products from Derris elliptica Benth’s root. They have included

the emulsifiable concentrates (EC), water milk and water-soluble powder preparations.

The technological protocol were established in many ways such as raw material

pre-processing treatment, extraction procedures, types of solvent used, stability of

rotenoids resin, biological activity, preservation and packaging. In fact, they have

successfully extracted rotenone from their native Derris species with the yield of

approximately 1.5 % (w/w) to 5.0 % (w/w) in dried roots using varies organic solvent

such as acetone, chloroform and ethanol on a batch solid-liquid of Normal Soaking

Extraction (NSE) method (Phan-Phuoc-Hien et al., 2003). Other processing parameters

(e.g.: solvent-to-solid ratio, raw material particles size, extraction temperature,

extraction duration, speed of agitation and etc.) used in the process are unknown but

generally ethanol is largely used as a solvent in the extraction process due to its low cost

and simple process (Hien-Phan-Phuoc et al., 1999). In Thailand, they have also

established the technological protocol on manufacturing the botanical insecticides. They

have also implemented a batch solid-liquid of Normal Soaking Extraction (NSE) method

with agitation under room temperature of 26 ± 2 0C and administered for 8 hours

(Suraphon and Manthana, 2001). Hence, they have managed to extract rotenone

approximately 5.2 % (w/w) in dried roots. According to Pitigon and Sangwanit (1997),

the most desirable solvent for the extraction of rotenone are ethanol and although

6

chloroform is also used as a solvent, it is proven to be dangerous to human health.

Therefore, ethanol is more suitable solvent for the extraction of rotenone in favour of

Thailand farmers. Unfortunately, rotenone based bio-pesticide manufacturer is

unavailable in Malaysia despite of its environmental-friendly effect and effectiveness to

treat the persistent insect pests of Diamondback moth (Plutella xylostella Linn.) that

always infested in the leafy vegetables farms. Thus, no rotenone based bio-pesticide

listed in the Pesticide Board of Malaysia registered products until May 2006. As for that

reason, Chemical Engineering Pilot Plant (CEPP) in Johor, Malaysia has initiated a

research on this particular active ingredient (rotenone) since 2001 and also being the

only research institute in Malaysia that undertake the research systematically by

commencing the selection of Derris species, pre-treatment, extraction, formulation,

laboratory bioassay and field trial, toxicity level and risk assessment until product

registration through the Pesticide Board of Malaysia (Saiful et al., 2003)

From all manufacturers and researchers that involved on producing the rotenone

as a potential botanical insecticides, they have one in common which is using a batch

solid-liquid extraction method even though they have implemented different processing

parameters and produced varies yield of rotenoids resin in dried roots; % (w/w) and

yield of rotenone in dried roots; % (w/w). In addition, not one of them has implemented

the biological activity (LC50) of brine shrimp (Artemia salina) to acquire rapid general

toxicity level in which correspond to the effect of processing parameter.

As for that reason, the objective of this research was to investigate the effect of

processing parameters (raw material particles size (mm in diameter), solvent-to-solid

ratio (ml/g) and types of solvent) on the yield and its biological activity (LC50) of

rotenone extracted from Derris elliptica using a batch solid-liquid extraction process.

7

1.2 Scopes of research

In order to achieve the objective, four scopes have been formulated in this

research. The scopes were:

(1) To investigate the effect of processing parameters on the yield of rotenoids resin

in dried roots; % (w/w).

(2) To investigate the effect of processing parameters on the yield of rotenone in

dried roots; % (w/w).

(3) To investigate the effect of processing parameters on the biological activity

(LC50) of brine shrimp (Artemia salina).

(4) To investigate the correlation between the biological activity (LC50) with the

yield of rotenoids resin in dried roots; % (w/w) and yield of rotenone in dried

roots; % (w/w).

The processing parameters studied were solvent-to-solid ratio (ml/g), types of

solvent and raw material particles size (mm in diameter). The other relevant parameters

involved in this research were fixed (control parameter) such as extraction temperature

(0C), weight of raw material (g) and extraction duration (hour). The experiments were

design using experimental design software called Design-Expert® software version 6.0

(Stat-Ease, 2002). Each data obtained from each run of experiments was evaluated for

the yield of rotenoids resin in dried roots; % (w/w), yield of rotenone in dried roots; %

(w/w) and biological activity (LC50) of rotenoids resin. Eventually, the correlation

between these response and independent variables were analyzed and interpreted.

8

1.3 Contribution of the research

This study contributes new knowledge in the area of phytochemical processing

and phytochemical pesticide:

(A) This research help to understand the main and interaction effects of the

processing parameters towards the yield of rotenoids resin, rotenone and their

biological activities (LC50). The correlation determined between independent

variables would further promote and enhance the usage of rotenoids resin as a

phytochemical pesticide or botanical insecticide products. Understanding the

effect of processing parameters against the yield of rotenoids resin, rotenone and

their biological activities (LC50) are essential in designing a better processing

technology to maintain and preserve the bio-active constituents in the extracts

and rotenoids resin effectively.

(B) Although Derris roots have been identified as a potential cash crop due to its

abundance growth in Malaysia, no research work of its own native species has

been conducted locally. The identification of appropriate processing parameters

to acquire maximum yield of rotenoids resin, rotenone and their biological

activities (LC50) against targeted and non-targeted organism are also have not

being studied. According to the local patent and industrial company database

related to the botanical insecticides production, there is no rotenone-based

industry listed in Malaysia until now. Therefore, the opportunities to develop an

option to the synthetic pesticides and environmental-friendly natural bio-

pesticide from local plant species are the main rationale why this extensive study

should be completed and carried out successfully.