VIVA-VOVE UTM 2006

SAIFUL IRWAN ZUBAIRI

Supervisor:Prof. Dr. Mohammad Roji Sarmidi

The Yield and Biological Activity of Rotenone Extracted From Derris elliptica

Presentation Outline

Introduction

Derris elliptica Background

Methodology

Results and Discussion

Conclusion

Introduction

Despite decades of warnings, the inappropriate use of chemical pesticides continues to pose threats to the environment and human health, especially in developing countries.

There have been massive upsurges in pesticide use in recent years (Harris, 1999), increasing use and often misuse has lead to increased problems of insecticide resistance.

IntroductionIn this research, the roots of Derris elliptica which is better known locally as Tuba plant is utilized as a raw material.

It is believed that the tuba plant contains an active ingredient known as Rotenone, which can be used to manufacture bio-pesticides.

Rotenone is a naturally occurring chemical with insecticidal and piscicidal properties extracted from the roots of several plant species belonging to genus Derris spp grown in Malaysia, East Indies and South America or Lonchocarpus spp grown in Central and South America .

IntroductionThis non-polar molecule, acts toxic towards cold-blooded animals but harmless to the man and environment. When exposed to sunlight, it is easily biodegrades to form Carbon Dioxide (CO2) & Water (H2O).

Low concentration in commercial products, degradability, and poor absorption across gut and skin in humans; are factors accounting for the good safety record of rotenone.

Rotenone has been approved by EPA as organic pesticide for organic farming due to low toxicity and low detectable residues in foods (Baker et. al., 2002).

Engineering Questions

What are critical process parameters?

How do we maximise yield?

What are the economically optimal operating conditions?

What are the processing parameters that reduce dissipation of the active ingredient throughout the whole extraction process?

How do we ensure active ingredient is present and in the correct amounts i.e. Standardisation

Objective & ScopesThe main objective of the study is to study the effect of processing parameters and the biological activity of rotenone extracted from Derris elliptica using a batch solid-liquid extraction process.

In order to achieve the objective, four scopes have been identified in this research. The scopes are:

To study the effect of processing parameters on the yield of rotenoids resin. To study the effect of processing parameters on the rotenone content. To study the effect of processing parameters on the biological activity.To study the interactions between the biological activity with the yield of rotenoids resin and the rotenone content.

Derris elliptica Background

Biology

Morphology

Phytochemistry

Analysis

Uses

Biology & Morphology

Biology Member of the Leguminosae Part of Fabaceae familyComposed of 200 genera and 68 species including 21 species of Tephrosia,12 of Derris, 12 of Lonchocarpus, 10 of Millettia and several of Mundula (John, 1944). Three species are found in Malaysia, which is Derris elliptica, Derris malaccensis and Derris uliginosa (Gaby Stroll, 1986).

MorphologySmall shrub originating in the tropical rainforestsGrows in lowland areas and does not thrive at higher altitudes Required 26 months for the maximum development of the rotenone.Derris can propagate vegetatively and fully developed after six weeks. Derris thrives on many soils but particularly on loams and clays (Gaby Stroll, 1986).

Phytochemistry

Active chemical constituentsIsoflavonoids - Major component is rotenone.

Rotenoids - consisted with all isoflavonoids.

Other Isoflavonoids - deguelin, elliptone, tephrosin, toxicarol,

malaccol & sumatrol (15-hydroxyrotenone).

Derris elliptica and Derris malaccensis contains 4 - 5 percent rotenone in dry roots.

Lonchocarpus utilis and Lonchocarpus urucu contain 8 - 10 percent rotenone in dry roots.

Phytochemistry

Rotenone molecular structure

Rotenone empirical formula C23H22O6

Molecular weight of 394.41 g/mol (Schnick, 1974)

Uses

TraditionallyUsed for fish poison, remedy for snake bites, skin sores, eczema and forehead relieve headaches.Root soaked or boiled with water and applied externally (Tinde Van Andel, 2000).

ScientificallySelective & non-systemic insecticide used on fruit crops and home gardens (WHO, 1992).For external treatment to treat tick, lice, scabies and other ectoparasites on pets (EXTOXNET, 1996).Fish eradications as part of water body management.

Analysis

Small number of compounds (<10 A.i)

Difficulty in identification and quantification if the photo-degradation occurred consequently decomposes to at least 20 degradation products (Cheng et al.1972).

Methods used include:

Thin Layer Chromatography

High Performance Liquid Chromatography

Compounds

Major Isoflavonoids that naturally occurred in derris resin (Rotenoids)

rotenone (m.p: 163 0C)

elliptone (m.p: 159 0C)

sumatrol/15-hydroxyrotenone (m.p: 188 0C)

malaccol/15-hydroxyelliptone (m.p: 244 0C)

toxicarol (m.p: 101 0C)

deguelin (m.p: 165 - 171 0C)

tephrosin/oxidation product of deguelin (m.p: 198 0C)


Methanol 95 %

Std Rf 0.63

Rf (8) 0.63

ROTENONE

Methanol, Under UV light 254 nm

ds

dc

8 7

6STD 9 10

ROTENONE

Methanol, Under UV light 365 nm

ds

dc

8 7

6STD 9 10

The presence of rotenone is identified under the UV light of 254 nm and 365 nm.

Solvent mixture of Petroleum Eter and Ethyl Acetate (4:2) – MOBILE PHASE.

CAMAG Linomat 5 (Semi Automatic Sampler Thin Layer Chromatography Device).

CAMAG LINOMAT 5

High Performance Liquid Chromatography (HPLC)

Based on Ronald L. Baron, (1976).

Using the external standard method for identification of rotenone content.

Using the Internal standard method for validation - OPTIONAL.

Rotenoids resin Calibration solution

Rotenone (6)

Tephrosin (5)

Deguelin (7)

PARAMETER SETTING

Column temperature Ambient

Flow rate 0.7 ml/min

UV Wavelength () 294 nm

Injection volume 5 µL

Methodology

PRELIMINARY PHASEPRELIMINARY PHASE

OPTIMIZATION PHASEOPTIMIZATION PHASE

VERIFICATION PHASEVERIFICATION PHASE

PRE-PROCESSINGPRE-PROCESSING

PROCESSING/EXTRACTIONPROCESSING/EXTRACTION

ANALYSIS OF RESPONSE VARIABLESANALYSIS OF RESPONSE VARIABLES

STATISTICAL ANALYSISSTATISTICAL ANALYSISPhases of the experiment

Flow diagram and overview of the project

There are four major steps involved in all three phases of experiments

Preliminary experimentsProcessing parameters of the extraction of rotenone at the preliminary experiments

Factor name Factor levels

Types of solvent Chloroform, Ethanol & Acetone

Solvent-to-solid ratio 10 ml/g & 3.3 ml/g

Raw material particles size Rough (5 – 2 mm) & Smooth (2 – 0.5 mm)

Extraction duration 0 – 1440 min (30 min interval times)

Fixed processing parameters of the extraction of rotenone at the preliminary experiments

Factor name Factor level

Weight of raw material 30 g of dried tuba roots

Extraction temperature Ambient temperature (26 0C – 30 0C)

Response variables of the extraction of rotenone

Response Variables Response Values

Yield of rotenone % (w/w)

Rotenone content mg (A.i)

Optimization experimentsBased on the preliminary study, an experimental design is developed based on the second-degree polynomial model of the response surface method (RSM).

3 factors out of 4 factors are chosen with 2 levels range which are the highest level (+α) & the lowest level (-α).

DESIGN EXPERT software version 6.0 (Stat-Ease. Inc., 2002) is used to design and interpret the significant effect of the experimental results.

The design of experiments used in the study is a Central Composite Design; CCD with 2nd Order Model - (23) with 30 runs including 3 centre point, 2 replicates & 1 alpha point (α).

TOTAL OF EXPERIMENT = [2n + 3 CP (Centre Point)] 2 Replicates] + [2n (α)] = (23 2 Replicates) + (3 CP 2 Replicates) + [ 23 1(α)]

= 16 + 6 + 8 = 30 experiments.

Processing parameters of the extraction of rotenoids resin at the optimization phase

Factor Factor name Factor levels

X1 Types of solvent Ethanol (-α) and Acetone (+α)

X2 Solvent-to-solid ratio 10 ml/g (-α) and 2 ml/g (+α)

X3 Raw material particles size 0.5 mm (-α) and 5 mm (+α)

Factor levels range is denoted as (α): +α = The highest level; -α = The lowest level

Fixed processing parameters of the extraction of rotenoids resin at the optimization phase

Factor name Factor level

Extraction duration Exhaustive extraction time (10 – 12 hours)

Weight of raw material 50 g of dried tuba roots

Extraction temperature Ambient temperature (26 0C – 30 0C)

Response variables of the extraction of rotenoids resin at the optimization phase

Response Variables Response Values

Yield of rotenoids resin mg (resin)

Rotenone content mg (A.i)

Brine Shrimp Lethality Bioassay Lethal Concentration (LC50) – mg/ml

Optimization experiments

Verification experiments

The verification phase is then carried out based on the results obtained from the optimization phase.

The experiments are carried out in 2 replicates.

The result obtained will verified the selection of the most appropriate processing parameters.

Various particles size of tuba roots

Rough (5 – 3 mm) Intermediate (2 – 1 mm) Smooth (1 – 0.5 mm)

Tuba Pre-processingAn important aspect of the phytochemical processing is the pre-processing of the herbal material prior to extraction.

For the preliminary phase, the procured tuba roots are sifted and separated into 2 main particles size, smooth (2 – 0.5 mm) and rough (5 – 2 mm) in diameter.

For the optimization phase, the procured tuba roots are sifted and separated accordingly to the particles size generated from the software.

Dried tuba roots in small pieces (Based on the particles size generated from the software)Dried tuba roots in small pieces (Based on the particles size generated from the software)

500 ml solvent (Acetone & Ethanol) added to 50 g sample – (10 mL/g) 500 ml solvent (Acetone & Ethanol) added to 50 g sample – (10 mL/g)

Normal Soaking Extraction for 10 - 12 hours (Ambient temperature) and stored in a dark placeNormal Soaking Extraction for 10 - 12 hours (Ambient temperature) and stored in a dark place

Filtered using Altech filter with GAST Laboratory Diaphragm Vacuum Pump & Compressor at 300 mbar.Filtered using Altech filter with GAST Laboratory Diaphragm Vacuum Pump & Compressor at 300 mbar.

Evaporated in rotary evaporator with vacuum pump at maximum vacuum of 300 mbar(Water bath heater temperature is set to be 40 0C for 1 hour – Remove 90 % of solvent)

Evaporated in rotary evaporator with vacuum pump at maximum vacuum of 300 mbar(Water bath heater temperature is set to be 40 0C for 1 hour – Remove 90 % of solvent)

Concentrated Liquid Crude Extract - Rotenoids resinConcentrated Liquid Crude Extract - Rotenoids resin

Store in a dark place at room temperature and avoid any light and excessive heat.Store in a dark place at room temperature and avoid any light and excessive heat.

Extraction of rotenoids resin from Tuba

Normal Soaking Extraction method

Construct the kinetic extraction curves

Content determination for 30 min interval

Analysis of Response Variables

PRODUCT ANALYSIS

Yield of rotenoids resin (% w/w)

Rotenone content (% w/w)

Lethal Concentration (LC50) of the biological activity analysis

µg/ml (10 mg/ml = 1 %)

High Performance Liquid Chromatography (Quantitative)


(Quantitative)

Product analysis is carried out on the response variables namely the yield of the extraction, rotenone content and the Lethal Concentration (LC50) of the biological activity analysis.

The eggs of brine shrimp, artemia salinas are readily available in pet shops.

Upon being placed in seawater, the eggs hatch within 48 hours to provide large numbers of larvae (nauplii) for experimental use

Allow 2 days for the shrimp to hatch and mature as naulpii.

Prepare vial for testing; for each fraction, test initially at 1000, 500, 100, 50, 10 g/ml; prepare 2 vials at each concentration for a total 10 vials plus 1 control vial.

Transfer 500 µL to vials corresponding to 1000, 500, 100, 50, 10 g/ml, respectively.

Rotenoids resin concentration is the initial concentration and dilution will be prepared accordingly to the amount of test concentration.

After 2 days (when the shrimp larvae are ready), add about 4 ml of seawater to each vial, count 10 shrimp per vial (50 shrimp/dilution) and adjust the volume with the seawater to 5 ml/vial.

Place the vials, uncovered and under the lamp. Be sure that the lamp does not overheat vials. 24 hours later count and record the number of survivors.

Brine Shrimp Lethality: A rapid General Bioassay for Bio-active Compounds

Lethal Concentration (LC50) of the biological activity analysis

Example of the dilution principles for preparing the bioassay concentration

1 ppm = 10-6 g/ml = 0.001 mg/ml

V0C0 = V1C1

(V0)(10) = (5)(1)V0 = 0.5 ml

V1C1 = V2C2

(V1)(1) = (5)(0.5)V1 = 2.5 ml

V2C2 = V3C3

(V2)(0.5) = (5)(0.1)V2 = 1.0 ml

V3C3 = V4C4

(V3)(0.1) = (5)(0.05)V3 = 2.5 ml

V4C4 = V5C5

(V3)(0.05) = (5)(0.01)V4 = 1 ml

Transfer 500 L of each fraction to the testing vial contains 10 shrimps/vial (5 ml/vial) for a replicate of 2 (2 fractions/vial)

70 ml 5 ml5 ml5 ml5 ml5 ml

Extract sample

C0 = 10.0 mg/ml

1000 ppm

(1.0 mg/ml)

500 ppm

(0.5 mg/ml)

100 ppm

(0.1 mg/ml)

50 ppm

(0.05 mg/ml)

10 ppm

(0.01 mg/ml)

500 L500 L500 L500 L500 L500 L500 L500 L500 L500 L

CONTROL

0.5 ml 2.5 ml 1.0 ml 2.5 ml 1.0 ml

Brine Shrimp Lethality: A rapid General Bioassay for Bio-active Compounds

Artemia salina mortality when exposed to extracts of the Derris elliptica

0 10 50 100 500 1000 Dose ((g/ml)

LC5050 %

100 %

Number of Mortality

Number of artemia salina mortality will be evaluated using the dose-response curves to determine the Lethal Concentration of rotenone that gives 50 % of the mortality.

Statistical AnalysisThe design of experiment (DOE) are used to evaluate the effects of several different factors on a response variable.

Statistical tool of (ANOVA) is used to analyze the data from the experiments and to make decisions about whether a given factor has a significant impact on the response variable.

In the optimization phase, the means of statistical analysis involves ANOVA and Response Surface Methodology (RSM).

The RSM is carried out based on the design of the experiments (DOE) generated by the Design Expert software version 6.0 (Stat-Ease Inc., 2000).

Contour plot for response surface and the optimum response can be built and the optimum parameters for each variables can be obtained from the response surface.

Preliminary Experiment Results

1.14

0.31

30.

692

0.60

2

0.28 0.

334

0.32

7

0.23

90.

111

0.12

5 0.22

5

0.06

3

0.13

4

0.18

1

0.15

3

0

0.2

0.4

0.6

0.8

1

1.2

Yie

ld o

f R

ote

no

ne

% (

w/w

) in

Liq

uid

C

rud

e E

xtra

ct o

f D

. elli

pti

ca

Kangkar Pulai(Fine Root)

Kangkar Pulai(Coarse

Root)

Kulai (Root) Kulai (Stem) Kangkar Pulai(Stem)

Sources of Sample

NORMAL SOAKING ACETONE

NORMAL SOAKING ETHANOL+H2O+OA

NORMAL SOAKING CHLOROFORM

Effects of the plant parts and types of solvent on yield

Yield of the Normal Soaking extraction method for different types of solvent


Graph: Kinetic equilibrium on the Rotenone extraction process

y = 0.2354Ln(x) - 0.0131

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 200 400 600 800 1000 1200 1400 1600

Time (min)

Ext

ract

ion

yie

ld (

% R

ote

no

ne

w/w

)

Graph: Kinetic equilibrium on the Rotenone extraction process

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 200 400 600 800 1000 1200 1400 1600

Time (min)

Ext

ract

ion

yie

ld (

% R

ote

no

ne

w/w

)

Extraction yield model and the effect of extraction duration on yield

Kinetic of Normal Soaking Extraction process of Derris elliptica roots: Yield of rotenone % (w/w)

Kinetic equilibrium of the rotenone extraction process (logarithmic)

Figure A Figure B


Graph of Rotenone Degradation vs Time of Concentration Process

35072

13002864 3005 2543

538 11033394

0

5000

10000

15000

20000

25000

30000

35000

40000

0 20 40 60 80 100 120

Time of Concentration Process (min)

Mas

s o

f R

ote

no

ne

(mg

)

Reduction of Bio-toxic constituent (Rotenone) duringconcentration process (Isothermal Process : 50 Degrees C)

Effects of the extraction temperature on yield

Degradation of rotenone occurred during the concentration process at 50 0C & 80 mbar of operating temperature and vacuum pressures respectively


Result of rotenone yield (mg/g dried Tuba roots)

Name of Samples mg/g dried Tuba roots

Tuba Kapur Kota Johor Lama (10.0 ml/g) 18.57


Result of rotenone concentration (mg/ml)

Name of Samples Concentration (mg/ml)



Result of rotenone content (mg)

Name of Samples Yield (mg)



Effect of the solvent-to-solid ratio on yield


Physical ParametersUV Absorbance - HPLC

Max: approximately 280 - 294 nm

Fraction of rotenone in dry roots1.14 % - 1.65 % of extract w/w: Acetone extract

Concentration of rotenone1.58 mg/mL - 2.87 mg/mL: Acetone extract

Mass of rotenone in dry roots285.07 mg - 870.00 mg: Acetone extract

Exhaustive extraction time 10 – 12 hours: Acetone extract

Preliminary Experiments Discussion

Longer duration leads to higher yield:

Max at 10 to 12 hours

52 % extracted within 30 min & 90% in 8 hrs

Acetone is the best solvent for obtaining the high rotenone content in fresh raw material as compared to the others two solvents (Chloroform & Ethanol).

Rotenone content in the root is always higher than in the stem.

Rotenone is being bio-accumulated at the root.

The initial high rate of extraction may be due to washing rather than the leaching process, where the bio-active compounds released from within cells by crushing or grinding are extensively quick on dissolving into the bulk solution.

The fine roots tend to be more superior in rotenone content because of the resin cell tissue that contains the rotenoids (Rotenone and its derivatives) are relatively abundant in roots of small and medium diameters (Francis A., 1943).

The best solvent-to-solid ratio for the Acetone extracts is 10 mL/g. In the optimization phase, the solvent-to-solid ratio of 10.0 mL/g & 2.0 mL/g will be used to obtain the optimum condition within the parameter values from the preliminary study and literature cited. Increasing the solvent-to-solid ratio increased the rotenone yield in the extraction. The increase of rotenone yield with the increase of the solvent-to-solid ratio is consistent with the mass transfer principles.

The solvent-to-solid ratio is strongly depends on the extraction conditions, by modifications on the solubility and solute-solvent interactions.

Expected Optimization Results

X: Types of solvent, Y: Solvent-to-solid ratioX: Raw material particles size, Y: Solvent-to-solid ratio

X: Raw material particles size, Y: Types of solvent

Z: Yield of rotenoids resin, rotenone content & biological activity

From the ANOVA table simulated by the software, the significant effects of each processing parameters (X,Y) towards the response variables (Z) will be calculated using the F-value and if the effects is significant, the optimum responses can be interpreted, obtained and the conclusion can be made conclusively. The optimum response variables will be summarized including the optimum processing parameters.

MAXIMUM YIELD OF ROTENOIDS RESIN = Q % (w/w)MAXIMUM ROTENONE CONTENT = R % (w/w)

MAXIMUM LETHAL CONCENTRATION (LC50) = J (µg/ml) = %(10 mg/ml = 1%)

The graphs consist of three axes where Z axes represent the response variable while X and Y axes represent three conditions as follows:

Expected Verification Results

Confirmation of the optimization.

Experiments will be carried out based on the most appropriate processing parameters obtained from the optimization experiments.

The results obtained verified the selection of the most appropriate processing parameters.

Response Variables Types ofsolvent

Solvent-to-solid ratio

Raw material particles size

RESULT (%)

Yield of rotenoids (% w/w) A1 B1 C1 D1

Rotenone content (% w/w) A2 B2 C2 D2

Bioassay LC50 (%) A3 B3 C3 D3

Expected results of yield, rotenone content and LC50 of the rotenoids resin based on the most appropriate processing parameters

Expected Comparison of the Optimum Response Variables

The optimum response variables obtained from the literature, preliminary, optimization and verification experiments will be compared and correlated to each other.

No literature is found for the Brine Shrimp Lethality (LC50) of rotenoids resin due to different method and systems of testing used by each researcher.

The analysis of rotenone content for the preliminary, optimization and verification phase will be used a standard method of analysis based on the method from Ronald L. Baron, (1976) and also from the AOAC Official Method, (1983).

Response variables Literature Preliminary Optimization Verification

Yield of rotenoids (% w/w) 39 %(SAPHYR, 1986)

E1 F1 G1

Rotenone content (% w/w) 14 %(SAPHYR, 1986)

E2 F2 G2

Bioassay LD50 (%) NA E3 F3 G3

Preliminary Experiments in Progress

Other preliminary work in progressFine tuning analysis method and apparatus.

Calibration of rotenone standard.

Determination of the effect of raw material particles size on yield.

Validation on the effect of the temperature on yield

The preliminary experiment result of raw material particles size will be used for the optimization experiments.

Conclusion

Future WorkDetermination of the effect of raw material particles size on yield – Preliminary experiment.

The optimization phase studies: effect of processing parameters on the response variables.

The verification phase studies: confirmation of the optimization.

Correlation between biological activity studies, yield of rotenoids resin and rotenone content.

Conclusion

RecommendationThe condition in which the Derris elliptica is collected play vital part in ensuring high rotenone content. It is believed that if the production of rotenone by Derris elliptica can be understood, the optimum derris plant can be cultivated and therefore increasing the overall yield of the extraction.

Rotenone dissipation during concentration process should be taken further noticed and yet there is not conclusive evidence to pronounce that by using the high temperature of operating condition can cause major losses of the toxicological constituents.

To perform an economic optimizations in order to determine an optimal economic process parameters.

The development of other processing techniques which reduced energy consumption, minimize degradation and maximise the yield of rotenone should be studied further.

QUESTION AND ANSWER SESSION

THANK YOU FOR YOUR KIND ATTENTION

NATURAL BIO-PESTICIDE LABORATORY ,

CEPP, UTM SKUDAI, JOHOR - 2004.

VIVA-VOVE UTM 2006

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